Synthetic nucleic acid molecule for imparting multiple traits

ABSTRACT

The present invention is directed to a DNA construct which includes a modified DNA molecule with a nucleotide sequence which is at least 80%, but less than 100%, homologous to two or more desired trait DNA molecules and which imparts the desired trait to plants transformed with the DNA construct. Each of the desired trait DNA molecules relative to the modified nucleic acid molecule have nucleotide sequence similarity values which differ by no more than 3 percentage points. The DNA construct may further include either a silencer or a plurality of modified DNA molecules. The present invention also relates to host cells, plant cells, transgenic plants, and transgenic plant seeds containing such DNA constructs. The present invention is also directed to a method of preparing a modified nucleic acid molecule suitable to impart multiple traits to a plant, a method of determining whether multiple desired traits can be imparted to plants by a single modified DNA molecule, and a method for imparting traits to plants by transforming the plants with the DNA construct.

This application is a divisional of U.S. patent application Ser. No.10/131,814, filed Apr. 24, 2002, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/286,075, filed Apr. 24, 2001,which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to DNA constructs containing a modifiednucleic acid molecule capable of imparting multiple desired traits to aplant, as well as host cells, plant cells, transgenic plants, andtransgenic plant seeds containing such DNA constructs. The presentinvention also relates to a method of preparing a modified nucleic acidmolecule capable of imparting multiple traits to a plant, a method ofdetermining whether multiple desired traits can be imparted to plants bya single modified DNA molecule, and a method for imparting traits toplants.

BACKGROUND OF THE INVENTION

Control of plant virus diseases took a major step forward when it wasshown that the tobacco mosaic virus (“TMV”) coat protein (“CP”) genethat was expressed in transgenic tobacco conferred resistance to TMV(Powell-Abel et al., “Delay of Disease Development in Transgenic Plantsthat Express the Tobacco Mosaic Virus Coat Protein Gene,” Science232:738-43 (1986)). The concept of pathogen-derived resistance (“PDR”),which states that pathogen genes that are expressed in transgenic plantswill confer resistance to infection by the homologous or relatedpathogens (Sanford et al., “The Concept of Parasite-DerivedResistance—Deriving Resistance Genes from the Parasite's Own Genome,” J.Theor. Biol., 113:395-405 (1985)) was introduced at about the same time.Since then, numerous reports have confirmed that PDR is a usefulstrategy for developing transgenic plants that are resistant to manydifferent viruses (Lomonossoff, G. P., “Pathogen-Derived Resistance toPlant Viruses,” Ann. Rev. Phytopathol., 33:32343 (1995)).

Remarkable progress has been made in developing virus resistanttransgenic plants despite a poor understanding of the mechanismsinvolved in the various forms of pathogen-derived resistance(Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,”Ann. Rev. Phytopathol. 33:323-43 (1995)). Although most reports dealwith the use of coat protein genes to confer resistance, a growingnumber of reports have shown that viral replicase (Golemboski et al.,“Plants Transformed with a Tobacco Mosaic Virus Nonstructural GeneSequence are Resistant to the Virus,” Proc. Natl. Acad. Sci. USA87:6311-15 (1990)), movement protein (Beck et al., “Disruption of VirusMovement Confers Broad-Spectrum Resistance Against Systemic Infection byPlant Viruses with a Triple Gene Block,” Proc. Natl. Acad. Sci. USA,91:10310-14 (1994)), NIa proteases of potyviruses (Maiti et al., “Plantsthat Express a Potyvirus Proteinase Gene are Resistant to VirusInfection,” Proc. Natl. Acad. Sci. USA, 90:6110-14 (1993)), and otherviral genes are effective. This led to the conclusion that any part of aplant viral genome may give rise to PDR. Furthermore, the viral genescan be effective in the translatable and nontranslatable sense forms,and less frequently, antisense forms (Baulcombe, “Mechanisms ofPathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell8:1833-44 (1996); Dougherty et al., “Transgenes and Gene Suppression:Telling us Something New?,” Current Opinion in Cell Biology 7:399-05(1995); Lomonossoff, G. P., “Pathogen-Derived Resistance to PlantViruses,” Ann. Rev. Phytopathol. 33:323-43 (1995)).

RNA-mediated resistance is the form of PDR where there is clear evidencethat viral proteins do not play a role in conferring resistance to thetransgenic plant. The first clear cases for RNA-mediated resistance werereported in 1992 for tobacco etch (“TEV”) potyvirus (Lindbo et al.,“Pathogen-Derived Resistance to a Potyvirus Immune and ResistancePhenotypes in Transgenic Tobacco Expressing Altered Forms of a PotyvirusCoat Protein Nucleotide Sequence,” Mol. Plant Microbe Interact. 5:144-53(1992)), potato virus Y (“PVY”) potyvirus (Van Der Vlugt et al.,“Evidence for Sense RNA-Mediated Protection to PVY in Tobacco PlantsTransformed with the Viral Oat Protein Cistron,” Plant Mol. Biol.,20:631-39 (1992), and for tomato spotted wilt (“TSWV”) tospovirus (deHaan et al., “Characterization of RNA-Mediated Resistance to TomatoSpotted Wilt Virus in Transgenic Tobacco Plants,” Bio/Technology10:1133-37 (1992)).

Other workers confirmed the occurrence of RNA-mediated resistance withpotyviruses (Smith et al., “Transgenic Plant Virus Resistance Mediatedby Untranslatable Sense RNAs: Expression, Regulation, and Fate ofNonessential RNAs,” Plant Cell 6:1441-53 (1994)), potexviruses (“PXV”)(Mueller et al., “Homology-Dependent Resistance: Transgenic VirusResistance in Plants Related to Homology-Dependent Gene Silencing,”Plant Journal 7:1001-13 (1995)), and TSWV and other tospoviruses (Panget al., “Resistance of Transgenic Nicotiana Benthamiana Plants to TomatoSpotted Wilt and Impatiens Necrotic Spot Tospoviruses: Evidence ofInvolvement of the N Protein and N Gene RNA in Resistance,”Phytopathology 84:243-49 (1994); Pang et al., “Different MechanismsProtect Transgenic Tobacco Against Tomato Spotted Wilt Virus andImpatiens Necrotic Spot Tospoviruses,” Bio/Technology 11:819-24 (1993)).More recent work has shown that RNA-mediated resistance also occurs withthe comovirus cowpea mosaic virus (Sijen et al., “RNA-Mediated VirusResistance: Role of Repeated Transgene and Delineation of TargetedRegions,” Plant Cell 8:2227-94 (1996)) and squash mosaic virus (Pang etal., “Resistance to Squash Mosaic Comovirus in Transgenic Squash PlantsExpressing its Coat Protein Genes,” Molecular Breeding 6:87-93 (2000)).

Major advances towards understanding the mechanism(s) of RNA-mediatedresistance were made by Dougherty and colleagues in a series ofexperiments with TEV and PVY. Using TEV, this group showed thattransgenic plants expressing translatable full length coat protein,truncated translatable coat protein, antisense coat protein genes, andnontranslatable coat protein genes had various phenotypic reactionsafter inoculation with TEV (Lindbo, J. A., “Pathogen-Derived Resistanceto a Potyvirus Immune and Resistant Phenotypes in Transgenic TobaccoExpressing Altered Forms of a Potyvirus Coat Protein NucleotideSequence,” Mol. Plant Microbe Interact. 5:144-53 (1992) and Lindbo etal., “Untranslatable Transcripts of the Tobacco Etch Virus Coat ProteinGene Sequence Can Interfere with Tobacco Etch Virus Replication inTransgenic Plants and Protoplasts,” Virology 189:725-33 (1992)).Transgenic plants displayed resistance, recovery (inoculated plantsinitially show systemic infection but younger leaves that develop laterare symptomless and resistant to the virus), or susceptible phenotypes.Furthermore, they showed that leaves of resistant plants andasymptomatic leaves of recovered plants had relatively low levels ofsteady state RNA when compared to those in leaves of susceptible plants(Lindbo et al., “Induction of a Highly Specific Antiviral State inTransgenic Plants: Implications for Regulation of Gene Expression andVirus Resistance,” Plant Cell 5:1749-59 (1993)). However, nuclear runoff experiments showed that those plants with low levels of steady stateRNA had higher transcription rates of the viral transgene than thoseplants that were susceptible (and had high steady state RNA levels). Toaccount for these observations, it was proposed “that the resistantstate and reduced steady state levels of transgene transcriptaccumulation are mediated at the cellular level by a cytoplasmicactivity that targets specific RNA sequences for inactivation” (Lindboet al., “Induction of a Highly Specific Antiviral State in TransgenicPlants: Implications for Regulation of Gene Expression and VirusResistance,” Plant Cell 5:1749-59 (1993)). It was also suggested thatthe low steady state RNA levels may be due to post-transcriptional genesilencing (“PTGS”), causing a lack of expression of the transcribedgene, a phenomenon that was first proposed by de Carvalho et al.,“Suppression of Beta-1,3-glucanase Transgene Expression in HomozygousPlants,” EMBO J. 11:2595-602 (1992) for the suppression ofβ-1,3-glucanase transgene in homozygous transgenic plants.

An RNA threshold model was proposed to account for these observations(Lindbo et al., “Induction of a Highly Specific Antiviral State inTransgenic Plants: Implications for Regulation of Gene Expression andVirus Resistance,” Plant Cell 5:1749-59 (1993)). This model states thatthere is a cytoplasmic cellular degradation mechanism that acts to limitthe RNA levels in plant cells, and that this mechanism is activated whenthe transgenic RNA transcript goes above a threshold level. Thedegradation mechanism is specific for the transcript that goes above thethreshold level; and if the transcript that goes above a certainthreshold is a viral transgene, the virus resistance state is observedin the plant, because the degradation mechanism also targets, forinactivation, the specific sequences of the incoming virus. The modelalso accounts for the ‘recovery’ of transgenic plants by suggesting thatviral RNA from the systemically invading virus triggers the phenomenonin some transgenic plants that have two copies of the transgenes. Plantsthat had more than three copies of the transgenes caused the thresholdlevel to be surpassed without the invasion of virus (Goodwin et al.,“Genetic and Biochemical Dissection of Transgenic RNA-Mediated VirusResistance,” Plant Cell 8:95-105 (1996); Smith et al., “Transgenic PlantVirus Resistance Mediated by Untranslatable Sense RNAs: Expression,Regulation, and Fate of Nonessential RNAs,” Plant Cell 6:1441-53(1994)). Although the degradation mechanism is not clear, it is proposedthat a cellular RNA dependent RNA polymerase (“RdRp”) binds to thetranscript and produces small fragments of antisense RNA which then bindto other transcripts to form duplexes which are then degraded bynucleases that specifically recognize RNA-RNA duplexes. This degradationmechanism is sequence specific, which accounts for the specificity ofRNA-mediated resistance.

Work on PVX by Baulcombe and colleagues (English et al., “Suppression ofVirus Accumulation in Transgenic Plants Exhibiting Silencing of NuclearGenes,” Plant Cell 8: 179-88 (1996); Mueller et al., “Homology-DependentResistance: Transgenic Virus Resistance in Plants Related toHomology-Dependent Gene Silencing,” Plant Journal 7:1001-13 (1995))confirmed and extended the results by Dougherty and colleagues. Anaberrant RNA model which is a modification of the RNA threshold model ofDougherty was proposed. The features of the model are similar to theDougherty model except that it states that the RNA level is not the soletrigger to activate the cellular degradation mechanism, but insteadaberrant RNAs that are produced during the transcription of thetransgene play an important part in activating the cytoplasmic cellularmechanism that degrades specific RNA. The production of aberrant RNA maybe enhanced by positional affects of the transgene on the chromosome andby methylation of the transgene DNA. The precise nature of the aberrantRNA is not defined, but it may contain a characteristic that makes it apreferred template for the production of antisense RNA by the hostencoded RdRp (Baulcombe, D. C., “Mechanisms of Pathogen-DerivedResistance to Viruses in Transgenic Plants,” Plant Cell 8:1833-44(1996); English et al., “Suppression of Virus Accumulation in TransgenicPlants Exhibiting Silencing of Nuclear Genes,” Plant Cell 8: 179-88(1996)). Thus, the model also proposes that RdRp and antisense moleculesare involved in the degradation mechanism. Baulcombe and colleaguesconfirmed that plants which show low steady state transgene levels havemultiple copies of transgenes and that the low steady state RNA and theaccompanying resistant state is due to post-transcriptional genesilencing. The term homology-dependent resistance was proposed todescribe the resistance in plants that show homology-dependent genesilencing (Mueller et al., “Homology-Dependent Resistance: TransgenicVirus Resistance in Plants Related to Homology-Dependent GeneSilencing,” Plant Journal 7:1001-13 (1995)).

Numerous reports have been published on critical advances in theunderstanding of the biochemistry and genetics of both gene silencingand RNA-interference. Similarities between RNA-interference (“RNAi”) andpost-transcriptional gene silencing are astonishing, and point all tothe crucial role played by sequence homology in triggering these twomechanistically related phenomena (Matzke et al., “RNA-Based SilencingStrategies in Plants,” Curr. Opin. Genet. Dev. 11(2):221-227 (2001)). InRNAi, the introduction of double stranded RNA into animal or plant cellsleads to the destruction of the endogenous, homologous mRNA,phenocopying a null mutant for that specific gene. In bothpost-transcriptional gene silencing and RNAi, the dsRNA is processed toshort interfering molecules of 21-, 22- or 23-nucleotide RNAs (“siRNA”)by a putative RNAaseIII-like enzyme (Tuschl T., “RNA Interference andSmall Interfering RNAs,” Chembiochem 2: 239-245 (2001); Zamore et al.,“RNAi: Double Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at21 to 23 Nucleotide Intervals,” Cell 101, 25-3, (2000)). Theendogenously generated siRNAs mediate and direct the specificdegradation of the target mRNA. In the case of RNAi the cleavage site inthe mRNA molecule targeted for degradation is located near the center ofthe region covered by the siRNA (Elbashir et al., “RNA Interference isMediated by 21- and 22-Nucleotide RNAs,” Gene Dev. 15(2):188-200(2001)).

Whether the same model applies for post-transcriptional gene silencingis still under debate (however, see Thomas et al., “Size Constraints forTargeting Post-Transcriptional Gene Silencing and for RNA-DirectedMethylation in Nicotiana Benthamiana Using a Potato Virus X Vector,”Plant J. 25(4):417-425 (2001)).

Tomato Spotted Wilt Virus (“TSWV”) is a very damaging virus of worldwidedistribution that attacks ornamentals and vegetable crops, causingmultimillion-dollar losses annually. TSWV belongs to the Tospoviridaefamily (Bunyavirus group), has a tripartite genome composed of sense andantisense RNA, and is transmitted by thrips in a persistent manner.Tobacco plants have been engineered to express full or partial versionof the nucleocapsid (“N”) gene of TSWV-BL. It has been clearlydemonstrated that any single fragment of the TSWV-BL N gene is able toconfer resistance against the virus by post-transcriptional genesilencing (Pang et al., “Nontarget DNA Sequences Reduce the TransgeneLength Necessary for RNA-Mediated Tospovirus Resistance in TransgenicPlants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997)). Moreover, thefragments can be reduced to a minimum of 100 nt long and still triggerpost-transcriptional gene silencing if transcriptionally fused to anon-related, carrier DNA (Pang et al., “Nontarget DNA Sequences Reducethe Transgene Length Necessary for RNA-Mediated Tospovirus Resistance inTransgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997) (“Pang1997)). Furthermore, it has been shown that the use of short fragmentsallowed the incorporation of viral gene fragments from multiple viralsources, imparting resistance to the plant against a plurality of viralpathogens (Jan, Doctor in Philosophy Thesis Dissertation, “Roles ofNon-Target DNA and Viral Gene Length in Influencing Multi-virusResistance Through Homology-Dependent Gene Silencing,” CornellUniversity, p. 286 (1988)). These short fragments, which individuallyhave insufficient length to impart such resistance, are more easily andcost effectively produced than full length genes. Furthermore, there isno need to include in the plant separate promoters for each of thefragments; only a single promoter is required.

Two important, and heretofore unanswered questions related togenetically engineered viral resistance using short viral DNA fragmentsare: 1) how do changes in sequence homology of the transgene affect itseffectiveness in conferring resistance, and 2) can a synthetic DNA beproduced with sufficient sequence homology such that the synthetic DNAwould confer resistance against multiple viruses. While great strideshave been made in PDR methodology, such as imparting resistance tomultiple viral pathogens, even Pang's method involves time-consuming andexpensive steps required to isolate and manipulate multiple viral DNAsfor transformation purposes. What is needed now is a method forutilizing sequence homology information to design and create a single,short synthetic transgene that will impart multiple traits, therebysignificantly reducing the labor and materials currently invested incloning and subcloning procedures directed to imparting pathogenresistance and other traits.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a DNA construct containing a modifiednucleic acid molecule with a nucleotide sequence which is at least 80%,but less than 100%, homologous to two or more desired trait DNAmolecules. Each of the desired trait DNA molecules, relative to themodified nucleic acid molecule, have a nucleotide sequences similarityvalue and each of these similarity values differs by no more than 3percentage points. The modified nucleic acid molecule imparts thedesired trait to plants transformed with the DNA construct.

The present invention also relates to a DNA construct containing aplurality of modified nucleic acid molecules, at least some of whichhave a nucleotide sequence which is at least 80%, but less than 100%,homologous to one or more desired trait DNA molecules. This plurality ofmodified nucleic acid molecules collectively impart their plurality oftraits to plants transformed with the DNA construct. At least some ofthe desired trait DNA molecules relative to their respective modifiednucleic acid molecule have a nucleotide sequence similarity value andeach of these similarity values differs by no more than 3 percentagepoints.

The present invention also relates to expression vectors, host cells,plant cells, transgenic plants, and transgenic plant seeds containingthe DNA construct of the present invention. Methods of imparting one ormore traits to plants with the DNA construct of the present inventionare also disclosed.

The present invention also relates to a method of preparing a modifiednucleic acid molecule suitable to impart multiple traits to a plant.This involves identifying a plurality of desired traits to be impartedto plants, where the desired traits are imparted by desired DNAmolecules having nucleotide sequences. This method also involvesselecting, as a reference nucleotide sequence, one nucleotide sequencefrom among the desired trait DNA molecules identified. The referencenucleic acid molecule is then modified to be at least 80%, but less than100%, homologous to the nucleotide sequences of the desired DNAmolecules identified. Each of the desired trait DNA molecules, relativeto the modified nucleic acid molecule, have a nucleotide sequencesimilarity value and each of these similarity values differs by 3percentage points or less.

The present invention also relates to a method of determining whethermultiple desired traits can be imparted to plants by a single modifiedDNA molecule. This involves identifying a plurality of desired traits tobe imparted to plants, where the desired traits are imparted by desiredtrait DNA molecules, having nucleotide sequences. One nucleotidesequence from among the desired trait DNA molecules identified isselected as a reference nucleotide sequence, and the referencenucleotide sequence is modified. A determination is then made whetherthe modified nucleic acid molecule is at least 80%, but less than 100%,homologous to the desired trait DNA molecules identified and whethereach of the desired trait DNA molecules, relative to the modifiednucleic acid molecule, have a nucleotide sequence similarity value andwhether each of these similarity values differs by no more than 3percentage points.

Post-transcriptional silencing influences traits (including viralresistance) in transgenic plants and its effect is sequence homologydependent. For example, a transgene will confer resistance to a virus ifit has a homology of 80% or greater to that virus. Applicants have takenadvantage of the sequence homology dependent characteristic ofpost-transcriptional silencing. Previously, it was shown that linking atrait DNA to a silencer DNA which triggers post-transcriptionalsilencing allows the use of trait DNA molecules that are shorter thanrequired if the trait DNA was used alone. For example, a 200 bp segmentof the N gene of TSWV does not confer resistance to transgenic plantswhen used alone as the transgene, but does confer resistance when linkedto a silencer DNA such as a green fluorescent protein. Thus, transgenicplants were obtained with multiple traits by linking short DNA traitmolecules to a silencer DNA. Two advantages of this previous finding arethat a single transgene with only one promoter could impart multipletraits, and that the length of the trait DNA could be considerablyshortened by linking them to a silencer DNA.

In accordance with the present invention, short synthetic DNAs canimpart multiple traits by simply making the synthetic DNA so that it hassufficient sequence homology to the individual DNA molecules to impartthe traits. For example, TSWV and groundnut ringspot virus (“GRSV”) aretwo tosposviruses that share only 76% homology within their N gene.Applicants synthesized a 216 bp DNA that has 90% homology to bothviruses and that DNA conferred resistance to both viruses when linked tothe silencer DNA. Compared to the previous finding: 1) this syntheticDNA was only 216 bp long (whereas, using the previous approach wouldneed to link a 216 bp segment of TSWV to a 216 bp segment of GRSV toimpart resistance to both viruses); 2) this synthetic DNA could easilybe synthesized without using any DNA from the virus as a template forgenerating the trait DNA whereas the latter had to be done when theprevious approach was used; 3) homology of the DNA to the trait DNAmolecules could be obtained by simply designing a synthetic DNA thatshowed at least 80% homology to the target trait molecules; and 4) thisinvention could be applied to any trait that is affected bypost-transcriptional silencing (and thus is sequence homologydependent). In short, synthetic genes that impart multiple traits can becustom designed by simply comparing nucleotide sequences of DNAs thatconfer separate traits and synthesizing a short DNA that has at least80% homology to each of the target trait DNAs. The number of traits canbe expanded by linking these synthetic DNA molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the scheme of the 216 bp TSWV fragment and thecorresponding primers to be named to be used for its synthesis. Thediagonal lines indicate the place in the oligonucleotides (“oligos”)where six random nucleotides and six nucleotides corresponding torestriction sites are located: XhoI for the 5′ end (left) and BamHI forthe 3′ end (right). The XX in the middle of the oligo name refers to thepercentage of change for every single construct according to Tables 3and 4. The fragment is shown in the sense orientation.

FIG. 2 is a schematic representation of the cloning/expression cassettevector pEPJ86GFP. The expression vector is located in a pUC backbonebetween the HindIII and KpnI sites in the multiple cloning site (MCS)adjacent to the lacZ gene. In the same order, from left to right, theexpression cassette contains a double 35S CaMV enhancer (the first two‘hatched’ regions), the 35S Cauliflower Mosaic Virus (CaMV) promoter(striped regions with thin vertical lines), the Alfalfa Mosaic Virusleader sequence (region with thick vertical lines), the greenfluorescent protein (GFP) gene (region of horizontal lines), and theCaMV 35S terminator sequence (bubbled region). After sequenceconfirmation the HindIII/KpnI fragments were subcloned into the planttransformation vector pGA482G.

FIG. 3 shows a gel electrophoresis of one of the PCR assembly andamplification products obtained in this work (e.g., 5% changes in thiscase). Row 1: a molecular ladder 100 bp step; Row 2: assembled productsafter 55 cycles of all mixed different oligonucleotides, as described inexamples; Row 3: a unique, single amplification product from theassembled reaction, prior to digestion.

FIGS. 4A-B show PCR analysis of some resulting transgenic lines. FIG. 4Ashows PCR analysis of the nptII gene; FIG. 4B shows PCR analysis of ¾ Ngene. Genomic DNA was isolated from independent transgenic lines asindicated. The examples shown here correspond to some plants transformedwith a gene with 5% changes scattered (5) or clustered at the 3′ end(5-3′). As a control, seed-derived plants from lines FJ-5 and FJ-22transformed with the native 3/3N gene were used, as well asnon-transgenic N. benthamiana DNA (C⁻). An additional positive control(C⁺) was DNA from the corresponding 5%-modified synthetic gene fragmentclones in pGA482G.

FIG. 5 shows susceptible and resistant plants: a transgenic (left) and anon-transgenic (right) plant four weeks after inoculation with TSWV-BL.

FIG. 6 shows a comparison of the ¾N gene of three differentTospoviruses: Tomato Spotted Wilt Virus (“TSWV”) (SEQ ID NO: 1),Groundnut Ringspot Virus (“GRSV”) (SEQ ID NO: 2), and Tomato ChloroticSpot Virus (“TCSV”) (SEQ ID NO: 3), and the Rec2 synthetic gene (SEQ IDNO: 4). Nucleotides that differ from Rec2 are shown in lower case.

FIG. 7 shows the starting sequences for the conserved regions of thecoat protein (CP) gene of the TH (SEQ ID NO: 6), KE (SEQ ID NO: 7) andYK (SEQ ID NO: 8) sequences from papaya ringspot virus compared to eachother and to the sequence of the modified synthetic nucleic acid (SEQ IDNO: 5) generated for targeting these PRSV isolates. The underlinedportions identify segments of more than 20 nt long of perfectsimilarity. The nucleotide changes differing from the synthetic sequenceare shown in lowercase.

FIG. 8 shows the distribution of dissimilar nucleotides compared to thesynthetic gene (SEQ ID NO: 9) for the variable regions of the of the CPgene of TH (SEQ ID NO: 10), KE (SEQ ID NO: 11) and YK (SEQ ID NO: 12).The underlined portions identify segments of more than 20 nt long ofperfect similarity. The nucleotide changes differing from the syntheticsequence are shown in lowercase.

FIG. 9 shows the CLUSTALW alignment of the nine selected potyvirussequences (SEQ ID NO: 13-SEQ ID NO: 22) and their consensus sequencebefore modification.

FIG. 10 shows the CLUSTALW alignment of the nine selected potyvirussequences and their consensus sequence from FIG. 9 after modification incomparison to the synthetic modified sequence (SEQ ID NO: 23).

FIG. 11 shows the CLUSTALW alignment of the five selected tomatopolygalacturonase sequences (SEQ ID NO: 24-SEQ ID NO: 29) beforemodification.

FIG. 12 shows the CLUSTALW alignment of the nine selected potyvirussequences and their consensus sequence from FIG. 11 after modificationin comparison to the synthetic modified potyvirus sequence (SEQ ID NO:30).

FIG. 13 shows the CLUSTALW alignment of the six selected Petuniachalcone synthase (“CHS”) sequences and their consensus sequence (SEQ IDNO: 31-SEQ ID NO: 37) before modification.

FIG. 14 shows the CLUSTALW alignment of the of the six selected Petuniachalcone synthase (“CHS”) and their consensus sequence from FIG. 13after modification in comparison to the synthetic modified Petunia CHSsequence (SEQ ID NO: 38).

FIG. 15 shows the CLUSTALW alignment of the seven selected Sorghum CHSgene sequences and their consensus sequence (SEQ ID NO: 39-SEQ ID NO:46) before modification.

FIG. 16 shows the CLUSTALW alignment of the seven selected Sorghum CHSgenes sequences and their consensus sequence from FIG. 15 aftermodification in comparison to the synthetic modified Sorghum CHSsequence (SEQ ID NO: 47).

FIG. 17 shows the CLUSTALW alignment of the nineteen selectedACC-oxidase (“ACC”) sequences from various plants and their consensussequence (SEQ ID NO: 48-SEQ ID NO: 67) before modification.

FIG. 18 shows the CLUSTALW alignment of the nineteen selectedACC-oxidase (“ACC”) sequences from various plants and their consensussequence from FIG. 17 after modification in comparison to the syntheticmodified ACC sequence (SEQ ID NO: 68).

FIG. 19 shows the CLUSTALW alignment of the resulting synthetic modified“Universal” PRSV isolate sequence in comparison with the startingsequences, the consensus sequences, and synthetic gene sequences (SEQ IDNO: 69-SEQ ID NO: 156).

FIG. 20 shows the CLUSTALW alignment of the resulting synthetic modified“Universal Americas” PRSV isolate sequence in comparison with thestarting sequences, the consensus sequence, and synthetic gene sequencefrom FIG. 19.

FIG. 21 shows the CLUSTALW alignment of the resulting synthetic modified“Universal Asia” PRSV isolate sequence in comparison with the startingsequences, the consensus sequence, and synthetic gene sequence from FIG.19.

FIG. 22 shows the CLUSTALW alignment of the resulting synthetic modified“Universal Pacific” PRSV isolate sequence in comparison with thestarting sequences, the consensus sequence, and synthetic gene sequencefrom FIG. 19.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a DNA construct containing a modifiednucleic acid molecule with a nucleotide sequence which is at least 80%,but less than 100%, homologous to two or more desired trait DNAmolecules. Each of the desired trait DNA molecules relative to themodified nucleic acid molecule have a nucleotide sequence similarityvalue and each of these similarity values differs by no more than 3percentage points. The modified nucleic acid molecule imparts thedesired trait to plants transformed with the DNA construct.

In one aspect of the present invention, the construct also includes asilencer DNA molecule operatively coupled to the modified nucleic acidmolecule. In this embodiment, the modified nucleic acid molecule andsilencer DNA molecule collectively impart the desired trait to plantstransformed with the DNA construct.

In another aspect of the present invention, the DNA construct can be afusion gene (chimera) which includes a plurality of modified DNAmolecules at least some of which have a nucleotide sequence that is atleast 80%, but less than 100%, homologous to the two or more desiredtrait DNA molecules. The plurality of trait molecules may be directed toone or more desired traits, and collectively impart the plurality oftraits to plants transformed with the DNA construct. At least some ofthe desired trait DNA molecules, relative to their respective modifiednucleic acid molecule, have a nucleotide sequence similarity value andeach of these similarity values differs by no more than 3 percentagepoints.

“Trait” as used herein refers to any characteristic geneticallydetermined in a given organism, as well as any characteristic that maybe imparted by transformation and regulated by post-transcriptional genesilencing. Therefore, the DNA molecules of the present invention may beendogenous (normally occurring), or exogenous (not normally occurring)in an organism.

The terms “homologous” and “homology” are used herein to mean astructural similarity between or among nucleic acid sequences, measuredas percent of similarity of corresponding deoxyribonucleotide bases (a,t, c, g) in two or more nucleic acid sequences compared to one anotherby any method. For example, once desired trait sequences are identified,they may be used to perform a BLAST search for further homologousnucleic acids in the GenBank database, or any other gene database. Forthe design, virtual assembly, and alignment of all synthetic and nativesequences, a computer program, such as DNAStar (LaserGene Software,Madison, Wis.), or the GeneDoc software ((Nicholas et al., “GeneDoc: ATool For Editing and Annotating Multiple Sequence Alignments” SoftwareVer. 2.6.002 (1997), and similarity trees can be generated with the TreeView software (Page, Software version 1.6. (2001), and both of theseprograms are freely available on the web and are suitable and herebyincorporated by reference in their entirety).

Percent similarity and percent identity are values used interchangeablyin the art as measures of nucleotide homology. For example, twosequences having perfect identity would have 100% similarity and,therefore, would be described as having perfect, or 100%, homology toone another.

One aspect of the present invention relates to the use of trait DNAmolecules which are exogenous to the plant, for example, DNA moleculesthat confer disease resistance to plants transformed with the DNAconstruct. The present invention is useful in plants for impartingresistance to a wide variety of pathogens including viruses, bacteria,fungi, viroids, phytoplasmas, nematodes, and insects. Resistance, interalia, to the following viruses can be achieved by the method of thepresent invention: tomato spotted wilt virus, impatiens necrotic spotvirus, groundnut ringspot virus, potato virus Y, potato virus X, tobaccomosaic virus, turnip mosaic virus, tobacco etch virus, papaya ringspotvirus, tomato mottle virus, tomato yellow leaf curl virus, arabis mosaicvirus, grapevine rupestris stem pitting associated virus-1, rupestrisstem pitting associated virus-1, grapevine leafroll-associated virus 3,grapevine leafroll-associated virus 4, grapevine leafroll-associatedvirus 8, grapevine leafroll-associated virus 1, grapevineleafroll-associated virus 5, grapevine leafroll-associated virus 7,grapevine leafroll-associated virus 2, grapevine virus A, grapevinetrichovirus B, grapevine virus B, or combinations thereof. Resistance,inter alia, to the following bacteria can also be imparted to plants inaccordance with present invention: Pseudomonas solancearum, Pseudomonassyringae pv. tabaci, Xanthomonas campestris pv. pelargonii, andAgrobacterium spp., including Agrobacterium tumefaciens. Plants can bemade resistant, inter alia, to the following fungi by use of the methodof the present invention: Fusarium oxysporum and Phytophthora infestans.Suitable DNA molecules include a DNA molecule encoding any gene of theviral genome, including a coat protein, a replicase, a DNA molecule notencoding protein, a DNA molecule encoding a viral gene product, orcombinations thereof. Furthermore, the present invention may also beused to impart genetic traits in many organisms, including, but notlimited to, bacteria, plants, and mammals.

The present invention is also used to confer traits other than diseaseresistance on plants. For example, DNA molecules which impart a genetictrait can be used as the desired trait molecule of the presentinvention. In this aspect of the present invention, suitable traitmolecules encode for desired color, enzyme production (or cessation ofproduction), plant hormones, or combinations thereof. Enzymes includethose involved in fruit development, such as ripening, or in anydevelopmental or metabolic pathway.

In one aspect of the present invention, the DNA construct of the presentinvention contains a plurality of modified trait DNA molecules operablylinked together. In this embodiment, each single modified trait moleculeis designed to impart a different collection of desired traits. For eachset of desired traits, a plurality of trait DNA molecules areidentified. From this plurality of trait DNA molecules, one is selectedas a reference sequence. This reference sequence is used to design amodified nucleic acid which is ultimately at least 80% (but less than100%) homologous to the trait DNA molecules. Each of these trait DNAmolecules, relative to the final modified nucleic acid molecule has anucleotide sequence similarity value and each of these similarity valuesdiffers by no more than 3 percentage points. This process may berepeated for other traits to be imparted to plants. For example, for“trait set 1” the desired trait to be imparted to plants is resistanceto both tomato spotted wilt virus (“TSWV”) and Groundnut Ringspot Virus(“GRSV”). For “trait set 1”, the DNA molecules which encode a coatprotein gene of TSWV and of GRSV will be individually identified andaligned to find a region of high homology (“homologized”). This region,or block of sequences of high homology is then modified to create asingle short nucleotide sequence with at least 80% homology to both ofthe identified DNA sequences (TSWV and GRSV).

This process may be repeated for additional desired traits (“trait set2,” “trait set 3,” etc.) to be imparted to plants, and the resultantmodified synthetic nucleic acid sequences for each trait set can belinked to prepare a fusion transgene, inserting, for example, the singlemodified synthetic nucleic acid molecule for “trait set 2” into thefusion gene vector behind the modified synthetic nucleic acid moleculefor “trait set 1.” In this embodiment, the modified nucleic acidmolecules collectively impart the desired trait(s) to plants transformedwith this nucleic acid construct.

In another aspect of the present invention, the fusion gene of thepresent invention also contains a “silencer”. This silencer DNA moleculeof the present invention can be selected from virtually any nucleic acidwhich effects gene silencing. The silencer molecule may be useful toenhance the triggering of RNA degradation effected by the syntheticmolecule of the present invention. This involves the cellular mechanismto degrade mRNA homologous to the transgene mRNA. The silencer DNAmolecule can be heterologous to the plant, need not interact with thetrait DNA molecule in the plant, and can be positioned 3′ to the traitDNA molecule. Examples of DNA molecules suitable as a silencer DNA inthe construct of the present invention include, without limitation, aviral cDNA molecule, a green fluorescence protein encoding DNA molecule,a plant DNA molecule, or combinations thereof. While not wishing to bebound by theory, is believed that in some situations, gene-silencing maybe more readily achieved when a silencer molecule is included in thetransgene. More particularly, the silencer DNA molecule is believed toboost the level of heterologous RNA within the cell above a thresholdlevel, contributing to post-transcriptional gene silencing. Thisactivates the degradation mechanism by which viral resistance (orconference of another trait) is achieved (Jan, F.-J., “Roles ofNontarget DNA and Viral Gene Length In Influencing Multi-VirusResistance Through Homology-Dependent Gene Silencing,” Faculty of theGraduate School, Cornell University, Ithaca, N.Y., p. 286 (1998); Jan etal., “A Minimum Length of N Gene Sequence in Transgenic Plants isRequired For RNA-Mediated Tospovirus Resistance,” J. General Virology81:235-242 (2000); Pang et al., “Nontarget DNA Sequences Reduce theTransgene Length Necessary For RNA-Mediated Tospovirus Resistance inTransgenic Plants,” Proc. Natl. Acad. Sci. USA 94:8261-8266 (1997),which are hereby incorporated by reference in their entirety).

In another aspect of the present invention, the silencer molecule isoperatively linked to a single modified trait nucleic acid molecule. Inthis construct, the single modified nucleic acid molecule has anucleotide sequence and length that is insufficient to impart its traitto plants transformed with the molecule; however, the modified DNAmolecule and the silencer molecule collectively impart the desiredtrait(s) to plants transformed with this DNA construct.

Alternatively, the construct of the present invention which contains aplurality of modified trait DNA molecules may also contain an operablylinked silencer molecule. In such a construct, each single modifiedtrait nucleic acid has a nucleotide sequence and length insufficient toimpart its trait(s) to the plant transformed with the molecule; however,the modified trait molecules and the silencer molecule collectivelyimpart the desired traits to the plant transformed with this construct.

Once a suitable modified trait DNA molecule is created, that nucleicacid molecule is incorporated into a DNA construct. This involvesinserting the modified homologized nucleic acid molecule into a vectorusing conventional recombinant DNA technology. Generally, this involvesinserting the nucleic acid molecule into an expression system to whichthe nucleic acid molecule is exogenous (i.e., not normally present). Theexogenous nucleic acid molecule is inserted into the expression systemwhich includes the necessary elements for the transcription andtranslation of the inserted protein coding sequences.

Preferably, the modified nucleic acid molecule of the present inventionis inserted in an expression system in such a way as to cause thetranscription of dsRNA in the host cell, to effect RNA-mediateddegradation and effect gene silencing. This can be accomplished in anumber of ways. One method involves further modifying the firstsynthetic nucleotide sequence using restriction enzyme-PCR methodologyto generate a second modified synthetic nucleotide sequence havinginverted nucleotide repeats and cloning the second modified nucleic acidinto an expression vector of choice. No special vector or furthermanipulation is required to achieve the dsRNA transcription. Anothersuitable method is to generate two identical molecules of a firstmodified nucleic acid and insert them into the expression vector withthe first copy in a forward-reverse fashion, i.e., one molecule facing5′→3′, and the second copy linked to the first and facing 3′→5′. The 5′promoter in such a construct can be at either end of the two linkedmolecules, as it will read through and transcribe a dsRNA of themodified synthetic nucleic acid molecule. Another suitable method is touse an expression vector specifically designed for making invertedrepeat transcripts of a gene, flanking a loop, which should efficientlyproduce a dsRNA. Exemplary binary vectors for this purpose include,without limitation, pMCG161 (Waterhouse et al., Proc. Nat'l Acad. Sci.USA 10(23):13959-64 (1998); Smith et al., “Total Silencing byIntron-Spliced Hairpin RNAs,” 407(6802): 319-320 (2000), which arehereby incorporated by reference in their entirety) and pFGC5941,pGSA1252, pGSA1204 and pGSA1285 (ChromDB, Univ. of AZ, Tucson, Ariz.).Other suitable vectors are those commercially available that aredesigned for dsRNA transcription or designed by the user of the presentinvention. Alternatively, the engineered self-annealing sequences can beengineered in such a way that they can be processed either by splicingin the nucleus (i.e., a single transgene that produces a singletranscript that might be processed post-transcriptionally) or bycytoplasmic enzymes to render dsRNAs only in this cellular compartment.New vectors have been developed elsewhere that might facilitate thistask (Wesley et al., “Construct Design For Efficient, Effective andHigh-Throughput Gene Silencing in Plants,” The Plant Journal 27: 581-590(2001), which is hereby incorporated by reference in its entirety).

The DNA constructs of the present invention also may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Suitable vectors include, butare not limited to, the following viral vectors such as lambda vectorsystem gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322,pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,pKC37, pKC101, SV 40, pBluescript II SK± or KS± (see “Stratagene CloningSystems” Catalog (1993) from Stratagene, La Jolla, Calif., which ishereby incorporated by reference in its entirety), pQE, pIH821, pGEX,pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase toDirect Expression of Cloned Genes,” Gene Expression Technology vol. 185(1990), which is hereby incorporated by reference in its entirety), andany derivatives thereof, as well as those mentioned above for producingdsRNA in the cell. Recombinant molecules can be introduced into cellsvia transformation, particularly transduction, conjugation,mobilization, or electroporation. The DNA sequences are cloned into thevector using standard cloning procedures in the art, as described bySambrook et al., Molecular Cloning: A Laboratory Manual Second Edition,Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., which are hereby incorporated by reference in their entirety.

In preparing a DNA vector for expression, the various DNA sequences maynormally be inserted or substituted into a bacterial plasmid. Anyconvenient plasmid may be employed, which will be characterized byhaving a bacterial replication system, a marker which allows forselection in a bacterium, and, generally, one or more unique,conveniently located restriction sites. Numerous plasmids, referred toas transformation vectors, are available for plant transformation. Theselection of a vector will depend on the preferred transformationtechnique and target species for transformation. A variety of vectorsare available for stable transformation using Agrobacterium tumefaciens,a soilborne bacterium that causes crown gall. Crown gall arecharacterized by tumors or galls that develop on the lower stem and mainroots of the infected plant. These tumors are due to the transfer andincorporation of part of the bacterium plasmid DNA into the plantchromosomal DNA. This transfer DNA (T-DNA) is expressed along with thenormal genes of the plant cell. The plasmid DNA, pTi, or Ti-DNA, for“tumor inducing plasmid,” contains the vir genes necessary for movementof the T-DNA into the plant. The T-DNA carries genes that encodeproteins involved in the biosynthesis of plant regulatory factors, andbacterial nutrients (opines). The T-DNA is delimited by two 25 bpimperfect direct repeat sequences called the “border sequences.” Byremoving the oncogene and opine genes, and replacing them with a gene ofinterest, it is possible to transfer foreign DNA into the plant withoutthe formation of tumors or the multiplication of Agrobacteriumtumefaciens (Fraley et al., “Expression of Bacterial Genes in PlantCells,” Proc. Nat'l Acad. Sci. 80:4803-4807 (1983), which is herebyincorporated by reference in its entirety).

Further improvement of this technique led to the development of thebinary vector system (Bevan, M., “Binary Agrobacterium Vectors for PlantTransformation,” Nucleic Acids Res. 12:8711-8721 (1984), which is herebyincorporated by reference in its entirety). In this system, all theT-DNA sequences (including the borders) are removed from the pTi, and asecond vector containing T-DNA is introduced into Agrobacteriumtumefaciens. This second vector has the advantage of being replicable inE. coli as well as A. tumefaciens, and contains a multiclonal site thatfacilitates the cloning of a transgene. An example of a commonly usedvector is pBin19 (Frisch, et al., “Complete Sequence of the BinaryVector Bin19,” Plant Molec. Biol. 27:405-409 (1995), which is herebyincorporated by reference in its entirety). Any appropriate vectors nowknown or later described for genetic transformation are suitable for usewith the present invention.

U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is herebyincorporated by reference in its entirety, describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase. These recombinant plasmidsare then introduced by means of transformation and replicated inunicellular cultures including procaryotic organisms and eukaryoticcells grown in tissue culture.

Certain “control elements” or “regulatory sequences” are alsoincorporated into the vector-construct. These include non-translatedregions of the vector, promoters, and 5′ and 3′ untranslated regionswhich interact with host cellular proteins to carry out transcriptionand translation. Such elements may vary in their strength andspecificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used.

A constitutive promoter is a promoter that directs expression of a genethroughout the development and life of an organism. Examples of someconstitutive promoters that are widely used for inducing expression oftransgenes include the nopoline synthase (NOS) gene promoter, fromAgrobacterium tumefaciens (U.S. Pat. No. 5,034,322 issued to Rogers etal., which is hereby incorporated by reference in its entirety), thecauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Pat. No.5,352,605 issued to Fraley et al., which is hereby incorporated byreference in its entirety), those derived from any of the several actingenes, which are known to be expressed in most cells types (U.S. Pat.No. 6,002,068 issued to Privalle et al., which is hereby incorporated byreference in its entirety), and the ubiquitin promoter, which is a geneproduct known to accumulate in many cell types.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed. The inducer can be achemical agent, such as a metabolite, growth regulator, herbicide, orphenolic compound, or a physiological stress directly imposed upon theplant such as cold, heat, salt, toxins, or through the action of apathogen or disease agent such as a virus or fungus. A plant cellcontaining an inducible promoter may be exposed to an inducer byexternally applying the inducer to the cell or plant such as byspraying, watering, heating, or by exposure to the operative pathogen.An example of an appropriate inducible promoter for use in the presentinvention is a glucocorticoid-inducible promoter (Schena et al., “ASteroid-Inducible Gene Expression System for Plant Cells,” Proc. Natl.Acad. Sci. 88:10421-5 (1991), which is hereby incorporated by referencein its entirety). Expression of the transgene-encoded protein is inducedin the transformed plants when the transgenic plants are brought intocontact with nanomolar concentrations of a glucocorticoid, or by contactwith dexamethasone, a glucocorticoid analog (Schena et al., “ASteroid-Inducible Gene Expression System for Plant Cells,” Proc. Natl.Acad. Sci. USA 88:10421-5 (1991); Aoyama et al., “AGlucocorticoid-Mediated Transcriptional Induction System in TransgenicPlants,” Plant J. 11: 605-612 (1997), and McNellis et al.,“Glucocorticoid-Inducible Expression of a Bacterial Avirulence Gene inTransgenic Arabidopsis Induces Hypersensitive Cell Death, Plant J.14(2):247-57 (1998), which are hereby incorporated by reference in theirentirety). In addition, inducible promoters include promoters thatfunction in a tissue specific manner to regulate the gene of interestwithin selected tissues of the plant. Examples of such tissue specificpromoters include seed, flower, or root specific promoters as are wellknown in the field (U.S. Pat. No. 5,750,385 issued to Shewmaker et al.,which is hereby incorporated by reference in its entirety). In thepreferred embodiment of the present invention, an exogenous promoter islinked to the nucleic acid of the construct, where “exogenous promoter”is defined as a promoter to which the nucleic acid of the construct isnot linked in nature.

The nucleic acid construct of the present invention also includes anoperable 3′ regulatory region, selected from among those which arecapable of providing correct transcription termination andpolyadenylation of mRNA for expression in the host cell of choice,operably linked to a modified trait DNA molecule of the presentinvention. A number of 3′ regulatory regions are known to be operable inplants. Exemplary 3′ regulatory regions include, without limitation, thenopaline synthase (“nos”) 3′ regulatory region (Fraley, et al.,“Expression of Bacterial Genes in Plant Cells,” Proc. Nat'l Acad. Sci.USA 80:4803-4807 (1983), which is hereby incorporated by reference inits entirety) and the cauliflower mosaic virus (“CaMV”) 3′ regulatoryregion (Odell, et al., “Identification of DNA Sequences Required forActivity of the Cauliflower Mosaic Virus 35S Promoter,” Nature313(6005):810-812 (1985), which is hereby incorporated by reference inits entirety). Virtually any 3′ regulatory region known to be operablein plants would suffice for proper expression of the coding sequence ofthe nucleic acid of the present invention.

The modified trait DNA molecule(s), a suitable promoter, silencing geneif included, and an appropriate 3′ regulatory region can be ligatedtogether to produce the expression systems which contain the DNAconstructs of the present invention, using well known molecular cloningtechniques as described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.(1989), and Ausubel et al. (1989) Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., which are herebyincorporated by reference in their entirety.

In any of the constructs of the present invention, the DNA moleculeimparting the desired trait can be positioned within the DNA constructin the sense (5′→3′) orientation. Alternatively, it can have anantisense (3′→5′) orientation. Antisense RNA technology involves theproduction of an RNA molecule that is complementary to the messenger RNAmolecule of a target gene. The antisense RNA can potentially block allexpression of the targeted gene. In the anti-virus context, plants aremade to express an antisense RNA molecule corresponding to a viral RNA(that is, the antisense RNA is an RNA molecule which is complementary toa plus sense RNA species encoded by an infecting virus). Such plants mayshow a slightly decreased susceptibility to infection by that virus.Such a complementary RNA molecule is termed antisense RNA.

It is possible for the DNA construct of the present invention to beconfigured so that the trait and silencer DNA molecules encode RNAmolecules which are translatable. As a result, that RNA molecule will betranslated at the ribosomes to produce the protein encoded by the DNAconstruct. Production of proteins in this manner can be increased byjoining the cloned gene encoding the DNA construct of interest withsynthetic double-stranded oligonucleotides which represent a viralregulatory sequence (i.e., a 5′ untranslated sequence) (U.S. Pat. No.4,820,639 to Gehrke, and U.S. Pat. No. 5,849,527 to Wilson, which arehereby incorporated by reference in their entirety).

Alternatively, the DNA construct of the present invention may beconfigured so that the modified trait and silencer molecules encode anmRNA which is not translatable, i.e., does not result in the productionof a protein or polypeptide. This is achieved, for example, byintroducing into the modified DNA sequence of the present invention oneor more premature stop codons, adding one or more bases (exceptmultiples of 3 bases) to displace the reading frame, and removing thetranslation initiation codon (U.S. Pat. No. 5,583,021 to Dougherty etal., which is hereby incorporated by reference in its entirety). Thiscan involve the use of a primer to which a stop codon, such as TAATGA,is inserted into the sense (or “forward”) PCR-primer for amplificationof the full nucleic acid, between the 5′ end of that primer, whichcorresponds to the appropriate restriction enzyme site of the vectorinto which the nucleic acid is to be inserted, and the 3′ end of theprimer, which corresponds to the 5′ sequence of the enzyme-encodingnucleic acid. Constructs containing nontranslatable DNA molecules may beparticularly useful for results which employ post-transcriptional genesilencing as a mechanism to achieve viral resistance in plantstransformed with the DNA constructs of the present invention.

Once the DNA construct of the present invention has been prepared, it isready to be incorporated into a host cell. Accordingly, another aspectof the present invention relates to a recombinant host cell containingone or more of the DNA constructs of the present invention. Basically,this method is carried out by transforming a host cell with a DNAconstruct of the present invention under conditions effective to yieldtranscription of the DNA molecule in the host cell, using standardcloning procedures known in the art, such as described by Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSprings Laboratory, Cold Springs Harbor, N.Y. (1989), which is herebyincorporated by reference in its entirety. Suitable host cells include,but are not limited to, bacteria, virus, yeast, mammalian cells, insect,plant, and the like. Preferably the host cells are either a bacterialcell or a plant cell. Methods of transformation may result in transientor stable expression of the DNA under control of the promoter.Preferably, a DNA construct of the present invention is stably insertedinto the genome of the recombinant plant cell as a result of thetransformation, to produce a heritable trait, although transientexpression can serve an important purpose, particularly when the plantunder investigation is slow-growing.

Plant tissue suitable for transformation include leaf tissue, roottissue, meristems, zygotic and somatic embryos, callus, protoplasts,tassels, pollen, embryos, anthers, and the like. The means oftransformation chosen is that most suited to the tissue to betransformed.

Transient expression in plant tissue is often achieved by particlebombardment (Klein et al., “High-Velocity Microprojectiles forDelivering Nucleic Acids Into Living Cells,” Nature 327:70-73 (1987),which is hereby incorporated by reference in its entirety). In thismethod, tungsten or gold microparticles (1 to 2 μm in diameter) arecoated with the DNA of interest and then bombarded at the tissue usinghigh pressure gas. In this way, it is possible to deliver foreign DNAinto the nucleus and obtain a temporal expression of the gene under thecurrent conditions of the tissue. Biologically active particles (e.g.,dried bacterial cells containing the vector and heterologous DNA) canalso be propelled into plant cells. Other variations of particlebombardment, now known or hereafter developed, can also be used.

An appropriate method of stably introducing the nucleic acid constructinto plant cells is to infect a plant cell with Agrobacteriumtumefaciens or Agrobacterium rhizogenes previously transformed with thenucleic acid construct. As described above, the Ti (or RI) plasmid ofAgrobacterium enables the highly successful transfer of a foreign DNAinto plant cells. Another approach to transforming plant cells with agene which imparts resistance to pathogens is particle bombardment (alsoknown as biolistic transformation) of the host cell, as disclosed inU.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford etal., and in Emerschad et al., “Somatic Embryogenesis and PlantDevelopment from Immature Zygotic Embryos of Seedless Grapes (Vitisvinifera),” Plant Cell Reports 14:6-12 (1995), which are herebyincorporated by reference in their entirety. Yet another method ofintroduction is fusion of protoplasts with other entities, eitherminicells, cells, lysosomes, or other fusible lipid-surfaced bodies(Fraley, et al., Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which ishereby incorporated by reference in its entirety). The DNA molecule mayalso be introduced into the plant cells by electroporation (Fromm etal., Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is herebyincorporated by reference in its entirety). In this technique, plantprotoplasts are electroporated in the presence of plasmids containingthe expression cassette. Electrical impulses of high field strengthreversibly permeabilize biomembranes allowing the introduction of theplasmids. Electroporated plant protoplasts reform the cell wall, divide,and regenerate. The precise method of transformation is not critical tothe practice of the present invention. Any method that results inefficient transformation of the host cell of choice is appropriate forpracticing the present invention.

After transformation, the transformed plant cells must be regenerated.Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. III(1986), and Fitch et al., “Somatic Embryogenesis and Plant Regenerationfrom Immature Zygotic Embryos of Papaya (Carica papaya L.),” Plant CellRep. 9:320 (1990), which are hereby incorporated by reference in itsentirety.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining explants is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these threevariables are controlled, then regeneration is usually reproducible andrepeatable.

Preferably, transformed cells are first identified using a selectionmarker simultaneously introduced into the host cells along with thenucleic acid construct of the present invention. Suitable selectionmarkers include, without limitation, markers encoding for antibioticresistance, such as the nptII gene which confers kanamycin resistance(Fraley, et al., Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), whichis hereby incorporated by reference in its entirety), and the geneswhich confer resistance to gentamycin, G418, hygromycin, streptomycin,spectinomycin, tetracycline, chloramphenicol, and the like. Cells ortissues are grown on a selection medium containing the appropriateantibiotic, whereby generally only those transformants expressing theantibiotic resistance marker continue to grow. Other types of markersare also suitable for inclusion in the expression cassette of thepresent invention. For example, a gene encoding for herbicide tolerance,such as tolerance to sulfonylurea is useful, or the dhfr gene, whichconfers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104(1983), which is hereby incorporated by reference in its entirety).Similarly, “reporter genes,” which encode for enzymes providing forproduction of a compound identifiable are suitable. The most widely usedreporter gene for gene fusion experiments has been uidA, a gene fromEscherichia coli that encodes the β-glucuronidase protein, also known asGUS. Jefferson et al., “GUS Fusions: β Glucuronidase as a Sensitive andVersatile Gene Fusion Marker in Higher Plants,'8 EMBO J. 6:3901-3907(1987), which is hereby incorporated by reference in its entirety.Similarly, enzymes providing for production of a compound identifiableby luminescence, such as luciferase, are useful. The selection markeremployed will depend on the target species; for certain target species,different antibiotics, herbicide, or biosynthesis selection markers arepreferred.

Plant cells and tissues selected by means of an inhibitory agent orother selection marker are then tested for the acquisition of the viralgene by Southern blot hybridization analysis, using a probe specific tothe viral genes contained in the given cassette used for transformation(Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Cold SpringHarbor, N.Y.: Cold Spring Harbor Press (1989), which is herebyincorporated by reference in its entirety).

After the DNA construct of the present invention is stably incorporatedin transgenic plants, the transgene can be transferred to other plantsby sexual crossing. Any of a number of standard breeding techniques canbe used, depending upon the species to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure so that theDNA construct is present in the resulting plants. Alternatively,transgenic seeds are recovered from the transgenic plants. These seedscan then be planted in the soil and cultivated using conventionalprocedures to produce transgenic plants.

The present invention can be utilized in conjunction with a wide varietyof plants or their seeds. Suitable plants include dicots and monocots.More particularly, useful crop plants can include: alfalfa, rice, wheat,barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato,bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet,parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion,garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini,cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry,pineapple, soybean, tobacco, tomato, sorghum, papaya, and sugarcane.Examples of suitable ornamental plants are: Arabidopsis thaliana,Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation,and zinnia.

Another aspect of the present invention is a method of imparting one ormore desired traits to plants. This may involve transforming a plantwith a DNA construct of the present invention containing a modifiedsynthetic nucleic acid molecule of the present invention, regeneratingthe transgenic plant, and propagating the progeny of the transgenicplant. Alternatively, the seed of the transgenic plant containing theconstruct of the present invention may be collected, and a transgenicplant propagated from the transgenic plant seed.

The present invention also relates to a method of preparing a modifiednucleic acid molecule suitable to impart multiple traits to a plant.This generally involves identifying a plurality of desired traits to beimparted to plants, where the desired traits are imparted by desiredtrait DNA molecules having nucleotide sequences. This method alsoinvolves selecting, as a reference nucleotide sequence, one nucleotidesequence from among the desired trait DNA molecules identified. Thenucleotide sequence of the reference nucleic acid molecule is thenmodified to form a modified nucleic acid molecule which is at least 80%homologous to the nucleotide sequences of the desired DNA moleculesidentified. Each of the desired trait DNA molecules, relative to themodified nucleic acid sequence, have a nucleotide sequence similarityvalue and each of these similarity values differs by 3 percentage pointsor less. This is an important attribute of the modified nucleic acidmolecule of the present invention. A synthetic nucleic acid moleculethat has a degree of homology within 3 percentage points of each desiredtrait DNA molecule can be expected to be an effector of RNA degradationin the host cell of a variety of plants. This is because the modifiednucleic acid molecule is more similar, on average, than many of thetarget starting sequences were to one another, or to the consensussequence. Therefore, it is the combined effect of a high similarityvalue (i.e., ≧80%) and a low range of variation in similarity relativeto the modified nucleic acid, that makes the synthetic molecule of thepresent invention an effective universal trigger of RNA degradation forthe desired trait molecules.

Trait nucleic acid sequences are obtained either from a public database,e.g., GenBank, from a commercial source, or may be known to those whointend to carry out this aspect of the present invention. Once desiredtrait sequences are identified, they may be used to perform a BLASTsearch for further homologous nucleotides at the GenBank database, orany other gene database. It is preferable that the nucleic acid sequenceselected as a trait molecule be selected from those known to encodefull-length, not partial mRNA, and areas of a genome including 5′ or 3′untranslated regions and introns be avoided.

Once the desired trait nucleic acid sequences are obtained, thepotential locations of sequences amenable to be manipulated to generatea short synthetic sequence are identified. This involves comparing thetrait sequences for sequence similarity (“homologizing”) to find acommon region of high homology among the trait sequences. Anycommercially or publicly available software can be used for analysis ofthe nucleotide sequences in this aspect of the present invention. Forexample, the comparative analysis of selected DNA sequences the DNAStarpackage (LaserGene Software, Madison, Wis.) can be used for aligning andediting. DNAStar and the Blast 2 Sequences from NCBI can be used tocompare sequences by pairs. To compare the sequence of interest with theGenBank database the Blast program is commonly used (Altschul et al.,“Gapped BLAST and PSI-BLAST: A New Generation of Protein Database SearchPrograms,” Nucleic Acid Research 25:3389-3402(1997), which is herebyincorporated by reference in its entirety). For multiple alignment, bothClustalW and Map are suitable (both of them freely available), whereasfor searching motifs MEME and MAST programs yield the best results(Bailey et al., “Family Pairwise Search with Embedded Motif Models,'8Bioinformatics 15(6):463-470 (1999); and Bailey et al., “Methods andStatistics for Combining Motif Match Scores,” J. Comput. Biol.5(2):211-221 (1998), which are hereby incorporated by reference in theirentirety). DNAStar is also capable of generating phylogenetic trees andsequence similarity matrixes. Alignments can also be analyzed with theGeneDoc software (Nicholas et al., “GeneDoc: A Tool For Editing andAnnotating Multiple Sequence Alignments” Software Ver. 2.6.002 (1997)and similarity trees generated with the Tree View software (Page,Software version 1.6. (2001) both of them freely available on the weband hereby incorporated by reference in their entirety).

A suitable length for the starting sequence of a homologized native DNA,in accordance with the present invention is in the range of 100-200 nts.By using small synthetic sequences, even in multiples, in a transgene,the amount of foreign DNA that is delivered to the plant is minimized.However, this is not meant in any way to set a minimum or maximum limiton the size of starting sequences or the size of the synthetic modifiednucleic acid sequence of the present invention. If two sequences arefound to be identical, one is discarded. If a region, or block ofsequences, of interest can be identified as having at least 80% but lessthan 100% homology, that block is chosen as the “starting sequence” foreach of the desired traits.

From this block of similar nucleotides, a single synthetic (meaning “notnaturally occurring”) nucleotide sequence with at least 80% homology anda low range of variation in similarity to each of the identified DNAsequences is formed. It is likely that a single synthetic sequence willnot be found that has the same percent similarity to each of thestarting sequence, and this is acceptable. However, the syntheticmodified sequence of the present invention will preferably not vary inpercent similarity by more than 3 percentage points when compared to theleast and most similar starting sequences. Even more preferably, thisvariation is no more than 2 percentage points, and most preferably, lessthan 1 percentage points. In theory, it is possible to create asynthetic sequence that has a 0% variation when compared to all startingsequences. Variations of 5% percentage points in the similarity valuesbetween each of the trait DNA molecules and the modified nucleic acidmolecule can also be useful.

The reference sequence used for modification to the final syntheticsequence may be any of the starting sequences or the consensus sequence.Preferably, the consensus sequence will not be chosen as the referencesequence when it contains any “N,” i.e., any unknown or unidentifiednucleotide in the alignment sequence, or when there is no consensus forthat position. Two other suitable choices are 1) the starting sequencemost similar to all other starting sequences (i.e., having the highestsimilarity value in comparison to all other trait nucleotides) or 2) thestarting sequence most dissimilar to all other starting sequences (i.e.,having the lowest similarity value in comparison to all other traitnucleotides). The process is the same regardless of the choice ofreference sequence. Nucleotide modifications are made step-wise withinthe reference sequence, changing those nucleotides that are mostdifferent from the other sequences to make the reference sequence moresimilar to the other starting sequences. The modifications made to thereference sequence will depend on the degree of similarity (or lackthereof) among the starting sequences, and between the startingsequences and the reference sequence. However, in no case is anindividual nucleotide that is 100% shared among the homologizedsequences modified. The reference sequence may, in some situations(e.g., when the consensus sequence or the most similar sequence, is usedas the reference sequence) need to be modified in such a way as todecrease the similarity between it and a potential final syntheticsequence in order to create a synthetic sequence that has a narrow rangeof variation when comparing the similarity of the synthetic sequence tothe least and most similar of the starting sequences. During themodification process, the reference sequence, having been modified as apotential synthetic sequence, will be analyzed against the otherstarting sequences to assess homology and range of homology. Thisprocess of modification of nucleotides followed by similarity analysismay need to be repeated numerous times to achieve a synthetic nucleotidesequence that has a narrow range of variation in similarity, if not 0%,to all the starting trait molecules. A “second” reference sequence canbe identified each time the process is repeated, so as to have a secondsequence to use for progressive comparative analysis during the process.The second reference sequence can be any of the trait sequences, theconsensus sequence from any of the comparisons, or it can be the samesequence chosen as the first reference sequence and previously modified.

When a modified synthetic nucleotide sequence is selected as having theattributes of a synthetic nucleic acid sequence of the present inventionand, therefore, is identified as suitable for use in imparting a trait,the synthetic nucleic acid may be prepared by recombinant methodologiesincluding, without limitation, site mutagenesis of one of the originaltrait nucleic acids; polymerase chain reaction, using an availableoriginal nucleic acid and suitable primers, or other methods of nucleicacid manipulation known in the art. As a preferable alternative, theidentified homologized nucleotide sequence of present invention can besynthesized using any DNA synthesizer capable of producing anoligonucleotide of the desired size. Thus, the present invention can becarried out without handling the actual source organism for the traitDNA. This eliminates the need for time-consuming recombinantmanipulations, significantly reducing both the effort and cost ofproducing a suitable modified nucleic acid sequence. The resultingmodified nucleotide is subsequently cloned into an expression vectorsuitable for transformation into a chosen host, as described above.

The present invention also relates to a method of determining whethermultiple desired traits can be imparted to plants by a single modifiedDNA molecule. This involves identifying a plurality of desired traits tobe imparted to plants, where the desired traits are imparted by desiredtrait DNA molecules having nucleotide sequences. One nucleotide sequencefrom among the desired trait DNA molecules identified is selected as areference nucleotide sequence, and the reference nucleotide sequence ismodified. A determination is then made regarding whether the modifiednucleic acid molecule is at least 80% homologous to the desired traitDNA molecules identified relative to the modified nucleic acid moleculeand whether each of these similarity values differs by no more than 3percentage points.

As noted above in the Background of the Invention, if sufficientsimilarity exists between siRNAs and the RNA target, degradation of theRNA ensues and gene silencing occurs. The determination of homologyamong the various trait nucleotide sequences may be made using anycomputer program capable of performing nucleotide homology analysis, forexample, the DNAStar alignment program. A modified DNA molecule which isdetermined to have an 80% or greater homology to each of the otheridentified trait nucleic acid sequences can be expected to impart thedesired trait to the transformed plant. The higher the homology over80%, the greater the expectation that modified nucleotide will impartthe desired multiple traits. In addition, the range of variability inhomology can be determined (by software analysis) that exists betweenthe modified trait DNA molecule and the nucleotide sequence of thefurther trait molecules. The less variation in the percent similarity(homology) to the synthetic sequence in comparison to all the startingsequences, the more efficient the DNA construct of the present inventionwill be in targeting all starting sequences for degradation.

This aspect of the present invention is illustrated in the generalizeddescription that follows.

Four nucleotide sequences having 20 nts each are used here to create asynthetic modified gene which is 80-100% homologous to each of the fourchosen nucleic acid sequences, in accordance with the present invention.Each of the starting sequences have only a narrow variation in homologyrelative to final synthetic sequence.

First, the desired nucleotide sequences are identified and obtained. Analignment is carried out to find a region of high homology among thetrait DNA sequences, which can be manipulated to create a syntheticsequence. This is done using any commercially available computersoftware. In this example, a region of 20 nucleotides was found to be alikely region for this purpose. These “starting sequences” are asfollows: Seq 1 ATAGCGTAGCTAGCTCGAGA (SEQ ID NO: 157) Seq 2CTGGCGTATCTAGGTCGAGA (SEQ ID NO: 158) Seq 3 ATAGCGTAACTAGCTCGAGA (SEQ IDNO: 159) Seq 4 ATGGCGTAGCTAGGTCGAGA (SEQ ID NO: 160)

Next, the homology, or degree of similarity, among the selected 20nucleotide starting sequences is determined by performing anotheralignment using a commercially available computer software. The resultsof the alignment for Seqs 1-4, showing nucleotide positions that aredifferent from among the sequences in boldfaced type, are as follows:Seq 1 ATAGCGTAGCTAGCTCGAGA (SEQ ID NO: 157) Seq 2 CTGGCGTATCTAGGTCGAGA(SEQ ID NO: 158) Seq 3 ATAGCGTAACTAGCTCGAGA (SEQ ID NO: 159) Seq 4ATGGCGTAGCTAGGTCGAGA (SEQ ID NO: 160) Consensus ATGGCGTAGCTAGGTCGAGA(SEQ ID NO: 161)

The percent of similarity by pairs determined by the alignment softwareis shown for the four starting nucleotides in Table 1, below. TABLE 1 12 3 4 1 75% 95% 90% 2 75% 90% 3 85%

The alignment information becomes the basis of the next step in theprocess of creating a synthetic nucleotide. Using the values ofsimilarity found by aligning the sequences, a single nucleotide sequenceis chosen as a reference sequence which will be manipulated to achievethe synthetic nucleotide. It may also be the consensus sequence (notshown here for Seq. 1-4) as ascertained by the alignment. It ispreferable, however, that the reference sequence be the sequence that ismost dissimilar from the other sequences.

Next, an approximation is made of how many nucleotides will need to bechanged to create the synthetic sequence. The initial assessment usesthe Formula as follows:Nucleotides to be changed=[(range of variation in homology)/2]×averagestarting sequence length Formula I

where the range of variation is taken from the alignment data. Here, forexample, the highest and lowest percentage of homology in the pair-wisealignment, shown in Table 1, above, are 95% and 75%, respectively, for atotal variation in homology 20%. Because one of the attributes of thesynthetic gene is a sequence with an even distribution of dissimilarity,the variation is divided by two, and then multiplied by the averagenumber of nucleotides in the starting sequences. Here, the averagestarting sequence number is 20 nt. Formula I is then applied as follows:[(95%-75%)/2×20 nt]=2 ntTherefore, the number of nucleotides that can be expected to be modifiedto create a synthetic gene of the present invention is approximatelytwo.

Nucleotide changes are then made in the reference sequence using thealignment charts to determine which changes will bring the referencesequence to a nucleotide composition that is within the goal of greaterthan 80%, and less than 100%, homology to all the starting sequences. Inthis example, Seq. 2 is a good choice as a reference sequence, becauseit has only a 75% similarity to Seq. 1 and Seq. 3, while all otherhomologies, pair-wise, are higher than 80%. To create a suitablesynthetic nucleotide sequence from the reference sequence, it ispreferable to avoid making a change to any nucleotide that is identicalin all sequences. A suitable starting point for making changes tonucleotides is to identify a position with a nucleotide that is sharedby more than two starting sequences, and change the reference sequenceto match at that position. For example, in Seq. 2, the referencesequence here, position 1 is “A” in three of the starting sequences, but“C” in Seq. 2. Therefore, modifying position 1 to an “A” in thereference sequence is the first change made here. Next, looking at thealignment data again, it is noted that in Seq. 2, the reference sequencehere, position 14 is “G.” Seq. 4 also has a “G” at position 14, whileSeq. 1 and 3 have “C” at position 14. Therefore, the starting sequenceof Seq. 2 is similar to one of the other four sequences at position 14,and dissimilar to two other sequences (25% similar overall). Changingthe “G” in position 14 of Seq. 2 to a “C” will raise the level ofhomology of the modified Seq. 2, i.e., the reference sequence above thatof the starting sequence of Seq. 2, because the reference sequence nowis similar at position 14 to 3 out of 5 sequences (the 4 startingsequences and the Reference seq now being a pool of 5), and dissimilarto 2 out 5 other sequences at that position (60% similar). In addition,a change to a “C” at position 14 makes “C” the nucleotide present at 75%of the sequences compared, as opposed to only 50% of the startingsequences.

Step-wise modifications in nucleotides are made in this fashion.Following the initial number of nucleotide changes in a given referencesequence suggested by the application of Formula I above, alignmentdeterminations are made to check if the modification(s) are bringing thereference sequence towards the goal of greater than 80, but less than100% homology in comparison to each of the starting sequences, andvarying in similarity by no more than 3% from each starting traitsequence. This process may need to be repeated a number of times forlarger sequences. Modifications can be considered complete when amodified sequence is determined to be at least 80, but less than 100%homologous to all the starting sequences, with the sequences also havinga narrow range of variance in percent homology (preferably 3% or less)among the starting sequences in comparison to the synthetic sequence.

For the four starting sequences in this example, the two modificationsmade at positions 1 and 14, respectively, resulted in the followingsynthetic sequence: Synthetic ATGGCGTATCTAGCTCGAGA (SEQ ID NO: 162)

The synthetic sequence compared to the starting sequences and theconsensus sequence for the starting sequences are below: Seq 1ATAGCGTAGCTAGCTCGAGA (SEQ ID NO: 157) Seq 2 CTGGCGTATCTAGGTCGAGA (SEQ IDNO: 158) Seq 3 ATAGCGTAACTAGCTCGAGA (SEQ ID NO: 159) Seq 4ATGGCGTAGCTAGGTCGAGA (SEQ ID NO: 160) Consensus ATGGCGTAGCTAGGTCGAGA(SEQ ID NO: 161) Synthetic ATGGCGTATCTAGCTCGAGA (SEQ ID NO: 162)Nucleotides that differ from the synthetic sequence are bolded. Thisexample also demonstrates that a classic consensus sequence is not thesame as the synthetic sequence of the present invention. Consensussequence software is designed to choose the nucleotide that is mostfrequently repeated in a given position when comparing two or moresequences. Because the synthetic sequence of the present inventionrequires not only high similarity, but a narrow range of variation, theconsensus sequence may vary (even considerably) from the synthetic geneof the present invention.

The homology of the final synthetic sequence compared to the startingsequences is shown in Table 2. TABLE 2 1 2 3 4 Cons. Sync. 1 75 95 90 9090 2 — 75 90 90 90 3 — 85 85 90 4 — 100  90 Cons. 90

In this illustration, each of the starting sequences has a similarityvalue of 90% in comparison to the modified sequence (i.e., each startingsequence differs from the modified nucleotide sequence by 2 out of 20).Therefore, the difference in the similarity value among the startingsequences relative to the modified sequence is 0%. This is an “ideal”modified nucleotide according to the present invention i.e., not only isthe degree of homology high relative to each starting sequence; each hasexactly the same degree of homology to the modified nucleic acid.

The universality of the present invention has been demonstrated by thefeasibility of inactivating genes by RNAi, not only in plants, but alsoin vertebrate and invertebrate systems (Caplen et al., “SpecificInhibition of Gene Expression By Small Double-Stranded RNAs inInvertebrate and Vertebrate Systems,” (2001)), including culturedmammalian cells (Elbashir et al., “Duplexes of 21-Nucleotides RNAsMediate RNA Interference In Cultured Mammalian Cells, Nature 411:494-498(2001), which are hereby incorporated by reference in their entirety).Thus, the present invention has potential application in manybiological, and even non-biological systems.

EXAMPLES Example 1 Synthetic Sequence Assembly and Amplification by PCR

The oligonucleotides used for the assembly and amplification of thesynthetic gene fragments derived from the ¾N gene of TSWV-BL are listedin Table 3, below. For the assembly and amplification of theaforementioned constructs the protocol of Stemmer et al., “Single-StepAssembly of a Gene and Entire Plasmid from Large Numbers ofOligodeoxyribonucleotides,” Gene 164(1):49-53 (1995), which is herebyincorporated by reference in its entirety, was essentially followed.Briefly, for the assembly step, 1 μl of each oligonucleotides (250 μM)corresponding to every single construct, were mixed in combinations asshown in Table 4 to produce the 10 final constructs. From this mixturean aliquot of 0.2 μl was mixed with 0.2 mM for each of the dNTPs, 50 mMKCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton® X-100, 2.25 mM MgCl₂, 1 unitof Taq polymerase and 0.5 units of Pfu polymerase in a final reactionvolume of 20 μl. Conditions for PCR, in all cases, were as follows: 55cycles of denaturing at 94° C. for 30 seconds, annealing at 52° C. for30 seconds, and extension at 72° C. for 30 seconds. From the assemblystep 2.5 μl were mixed for amplification with the appropriate pair ofprimers, as shown in Table 2, at a final concentration of 1 μM each, 0.2mM for each of the dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 %Triton® X-100, 2.25 mM MgCl₂, 5 units of Taq polymerase and 0.5 units ofPfu polymerase in a final reaction volume of 100 μl. Conditions foramplification were: 35 cycles of denaturing at 92° C. for 30 seconds,annealing at 65° C. for 30 seconds and extension at 72° C. for 2minutes. Size and yield of every single product were determined byagarose gel electrophoresis with molecular markers of knownconcentration. TABLE 3 Oligos used for the synthesis and amplificationof all different constructs SEQ ID NO: 163:5′-ATCATTCTCGAGGCAAAGTCTGTGAGGC-3′ TS-N-1AH SEQ ID NO: 164:5′-TTGCCATAATGCTGGGAGGTAGCTTACCTCTTATTGCTTC-3′ TS-N-2H SEQ ID NO: 165:5′-AGTTGATAGCTTTGAGATGATCAGTGTTGTCTTGGCTATA-3′ TS-N-3H SEQ ID NO: 166:5′-TATCAGGATGCAAAATACAAGGATCTCGGGATCGACCCAA-3′ TS-N-4H SEQ ID NO: 167:5′-AGAAGTATGACACCAGGGAAGCCTTAGGAAAAGTTTGCAC-3′ TS-N-5H SEQ ID NO: 168:5′-TGTGCTGAAAAGAAAAGCATTTGAAATGAATGAAGATCAG-3′ TS-N-6H SEQ ID NO: 169:5′-AGCTAAGGATCCCTGATCTTCATTCATTTCAA-3′ TS-N-1AC SEQ ID NO: 170:5′-ATGCTTTTCTTTTCAGCACAGTGCAAACTTTTCCTAAGGC-3′ TS-N-2C SEQ ID NO: 171:5′-TTCCCTGGTGTCATACTTCTTTGGGTCGATCCCGAGATCC-3′ TS-N-3C SEQ ID NO: 172:5′-TTGTATTTTGCATCCTGATATATAGCCAAGACAACACTGA-3′ TS-N-4C SEQ ID NO: 173:5′-TCATCTCAAAGCTATCAACTGAAGCAATAAGAGGTAAGCT-3′ TS-N-5C SEQ ID NO: 174:5′-ACCTCCCAGCATTATGGCAAGCCTCACAGACTTTGCCTCG-3′ TS-N-6C SEQ ID NO: 175:5′-TTCCCATAATGCTGGGAGGTATCTTACCTCTTATTGCTTC-3′ TS-5-2H SEQ ID NO: 176:5′-TGTTGATAGCTTTGAGATGTTCAGTGTTGTCTTGGCTAAA-3′ TS-5-3H SEQ ID NO: 177:5′-TATCAGGATGCAAAATAGAAGGATCTCGGGATCGACGCAA-3′ TS-5-4H SEQ ID NO: 178:5′-AGAAGTATGACACCACGGAAGCCTTAGGAAAAGTATGCAC-3′ TS-5-5H SEQ ID NO: 179:5′-TGTGCTGAAAAGATAAGCATTTGAAATGAATGTAGATCAG-3′ TS-5-6H SEQ ID NO: 180:5′-AGCTAAGGATCCCTGATCTACATTCATTTCAA-3′ TS-5-1AC SEQ ID NO: 181:5′-ATGCTTATCTTTTCAGCACAGTGCATACTTTTCCTAAGGC-3′ TS-5-2C SEQ ID NO: 182:5′-TTCCGTGGTGTCATACTTCTTTGCGTCGATCCCGAGATCC-3′ TS-5-3C SEQ ID NO: 183:5′-TTCTATTTTGCATCCTGATATTTAGCCAAGACAACACTGA-3′ TS-5-4C SEQ ID NO: 184:5′-ACATCTCAAAGCTATCAACAGAAGCAATAAGAGGTAAGAT-3′ TS-5-5C SEQ ID NO: 185:5′-ACCTCCCAGCATTATGGGAAGCCTCACAGACTTTGCCTCG-3′ TS-5-6C SEQ ID NO: 186:5′-ATCATTCTCGAGCGTTTCAGACAGAGGC-3′ TS-5-5′-1AH SEQ ID NO: 187:5′-ACCTCCCAGCATTATGGCAAGCCTCTGTCTGAAACGCTCG-3′ TS-5-5′-6C SEQ ID NO:188: 5′-TGTGCTGAAAAGAAAAGCATTTGAAATGATACTTCTAGTC-3′ TS-5-3′-6H SEQ IDNO: 189: 5′-AGCTAAGGATCCGACTAGAAGTATCATTTCAA-3′ TS-5-3′-1AC SEQ ID NO:190: 5′-ATAGTCCTACGAAAATACAAGGATCTCGGGATCGACCCAA-3′ TS-5-m-4H SEQ ID NO:191: 5′-TTGTATTTTCGTAGGACTATTATAGCCAAGACAACACTGA-3′ TS-5-m-4C SEQ ID NO:192: 5′-ATCATTCTCGAGCGTTTCAGACACTCCG-3′ TS-10-5′-1AH SEQ ID NO: 193:5′-AACATAAAACGTAGGACTATTATAGCCAAGACAACACTGA-3′ TS-10-m-4C SEQ ID NO:194: 5′-AGCTAAGGATCCGACTAGAAGTAAGTAAAGTT-3′ TS-10-3′-1AC SEQ ID NO: 195:5′-ATCATTCTCGAGCCAAAGACTGTGACGC-3′ TS-15-1AH SEQ ID NO: 196:5′-TTGCGATAATGGTGGGAGCTAGCTTTCCTCTTTTTGCTTG-3′ TS-15-2H SEQ ID NO: 197:5′-AGTTGACAGCTTTCAGATGAACAGTGTAGTCTTGCCTATA-3′ TS-15-3H SEQ ID NO: 198:5′-TTTCAGGAAGCAAAAAACAAGGTTCTCGGCATCGACGCAA-3′ TS-15-4H SEQ ID NO: 199:5′-AGATGTATGAGACCAGGCAAGCCTAAGGAAATGTTTGCTC-3′ TS-15-5H SEQ ID NO: 200:5′-TGTGCTCAAAAGATAAGCATATGAAATCAATGAACATCAC-3′ TS-15-6H SEQ ID NO: 201:5′-AGCTAAGGATCCGTGATGTTCATTGATTTCAT-3′ TS-15-1AC SEQ ID NO: 202:5′-ATGCTTATCTTTTGAGCACAGAGCAAACATTTCCTTAGGC-3′ TS-15-2C SEQ ID NO: 203:5′-TTGCCTGGTCTCATACATCTTTGCGTCGATGCCGAGAACC-3′ TS-15-3C SEQ ID NO: 204:5′-TTGTTTTTTGCTTCCTGAAATATAGGCAAGACTACACTGT-3′ TS-15-4C SEQ ID NO: 2055′-TCATCTGAAAGCTGTCAACTCAAGCAAAAAGAGGAAAGCT-3′ TS-15-5C SEQ ID NO: 206:5′-AGCTCCCACCATTATCGCAAGCGTCACAGTCTTTGGCTCG-3′ TS-15-6C SEQ ID NO: 207:5′-ACCTGGGTCGTAATACCGTTCGGAGTGTCTGAAACGCTCG-3′ TS-15-5′-6C SEQ ID NO:208: 5′-TGTGCTGATTTCTTTTCGTAAACTTTACTTACTTCTAGTC-3′ TS-15-3′-6H SEQ IDNO: 209: 5′-TACGAAAAGAAATCAGCACAGTGCAAACTTTTCCTAAGGC-3′ TS-15-3′-2C SEQID NO: 210: 5′-ATAGTCCTACGTTTTATGTTCCTAGAGCCCTACGACCCAA-3′ TS-15-m-4HSEQ ID NO: 211: 5′-TTCCCTGGTGTCATACTTCTTTGGGTCGTAGGGCTCTAGG-3′TS-15-m-3C SEQ ID NO: 212: 5′-ATCATTCTCGAGGCAATGTCTCTGAGCC-3′ TS-20-1AHSEQ ID NO: 213: 5′-TTGGCATATTGCTCGGAGCTAGCATACCACTTAATGCTAC-3′ TS-20-2HSEQ ID NO: 214: 5′-AGTAGATACCTTTCAGATCATCACTGTTCTCTTCGCTAAA-3′ TS-20-3HSEQ ID NO: 215: 5′-TATGAGGAAGCAATATACTAGGAACTCGCGATCCACCCTA-3′ TS-20-4HSEQ ID NO: 216: 5′-AGATGTATCACACGAGGGTAGCCATAGGTAAAGATTGCTC-3′ TS-20-5HSEQ ID NO: 217: 5′-TGTCCTGATAAGATAAGCTTTTGTAATGTATGATGATCTG-3′ TS-20-6HSEQ ID NO: 218: 5′-AGCTAAGGATCCCAGATCATCATACATTACAA-3′ TS-20-1AC SEQ IDNO: 219: 5′-AAGCTTATCTTATCAGGACAGAGCAATCTTTACCTATGGC-3′ TS-20-2C SEQ IDNO: 220: 5′-TACCCTCGTGTGATACATCTTAGGGTGGATCGCGAGTTCC-3′ TS-20-3C SEQ IDNO: 221: 5′-TAGTATATTGCTTCCTCATATTTAGCGAAGAGAACAGTGA-3′ TS-20-4C SEQ IDNO: 222: 5′-TGATCTGAAAGGTATCTACTGTAGCATTAAGTGGTATGCT-3′ TS-20-5C SEQ IDNO: 223: 5′-AGCTCCGAGCAATATGCCAAGGCTCAGAGACATTGCCTCG-3′ TS-20-6C SEQ IDNO: 224: 5′-ATCATTCTCGAGGCAAAATCTGTGAGAC-3′ Rec2-1AH SEQ ID NO: 225:5′-TTGCCATAATGCTGGGAGGTAGTATCCCTCTTATTGCTTC-3′ Rec2-2H SEQ ID NO: 226:5′-TGTTGACAGCTTTGAAATGATCAGTGTTGTCCTTGCTATA-3′ Rec2-3H SEQ ID NO: 227:5′-TATCAAGATGCAAAATACAAGGATCTCGGGATTGAACCAA-3′ Rec2-4H SEQ ID NO: 2285′-CGAAGTATAACACTAAGGAAGCCTTAGGAAAAGTTTGCAC-3′ Rec2-5H SEQ ID NO: 2295′-TGTGCTGAAAAGCAAAGGATTTACAATGGATGAAGATCAG-3′ Rec2-6H SEQ ID NO: 2305′-AGCTAAGGATCCCTGATCTTCATCCATTGTAA-3′ Rec2-1AC SEQ ID NO: 2315′-ATCCTTTGCTTTTCAGCACAGTGCAAACTTTTCCTAAGGC-3′ Rec2-2C SEQ ID NO: 2325′-TTCCTTAGTGTTATACTTCGTTGGTTCAATCCCGAGATCC-3′ Rec2-3C SEQ ID NO: 2335′-TTGTATTTTGCATCTTGATATATAGCAAGGACAACACTGA-3′ Rec2-4C SEQ ID NO: 2345′-TCATTTCAAAGCTGTCAACAGAAGCAATAAGAGGGATACT-3′ Rec2-5C SEQ ID NO: 2355′-ACCTCCCAGCATTATGGCAAGTCTCACAGATTTTGCCTCG-3′ Rec2-6C

FIG. 1 shows the scheme of the 216 bp TSWV fragment and thecorresponding primers to be named to be used for its synthesis. Thediagonal lines indicate the place in the oligos where six randomnucleotides and six nucleotides corresponding to restriction sites arelocated: XhoI for the 5′ end (left) and BamHI for the 3′ end (right).The XX in the middle of the oligo name refers to the percentage ofchange for every single construct according to Tables 1 and 2. Thefragment is shown in the sense orientation.

Example 2 Cloning and Sequencing

All gene fragments were digested with an excess of BamHI and XhoI for noless than 12 hours, according to Sambrook et al., “Molecular Cloning: ALaboratory Manual,” Cold Spring Harbor, N.Y.: Cold Spring Harbor Press(1989), which is hereby incorporated by reference in its entirety. Thedigested fragments were excised from agarose gels and column purifiedfor ligation into the BamHI/XhoI cloning site of vector pEPJ86GFP, shownin FIG. 2. In this cloning vector, a transcriptional fusion with GFPgene was created under the control of a double 35S promoter. Forchemical or electro transformation either Escherichia coli DH5α orXL1-Blue were routinely used. For plasmid miniprep either the alkalinelysis or the boiling method were used. Five independent recombinantplasmids per construct were sequenced; plasmids with identical sequenceto the computer-generated gene fragments were chosen for digestion andsubsequent subcloning into pGA482G. Digestion with KpnI and HindIIIrenders a subcloning fragment with the GFP gene fused to the synthetic ¾N gene fragment, and under the control of the 35S promoter. Afterchecking by PCR and restriction analysis the recombinant derivatives ofpGA482G were sequenced again before transforming Agrobacteriumtumefaciens LBA4404. A. tumefaciens transformants were kept at −80° C.after checking for the recombinant plasmid by restriction analysis. PCRproducts from transgenic plants were also column purified and sequencedfrom at least three different transgenic lines. All sequencing was doneusing an ABI 373 automated sequencer.

Example 3 Plant Transformation

Leaf discs of young, greenhouse-grown Nicotiana benthamiana plants weretransformed with A. tumefaciens as described essentially by Horsch(Horsch et al., “A Simple and General Method for Transferring Genes intoPlants,” Science, 227:1229-31 (1985), which is hereby incorporated byreference in its entirety). Transformed leaf discs were selected andregenerated in MS medium containing 250 mg/L of carbenicillin and 150mg/L of kanamycin sulfate. Rooting was induced in a hormone-free mediumin the presence of both antibiotics. All regenerated and rooted explantsderived from independent calli. Fully rooted transformants weretransferred to Cornell soil mixture and grown under greenhouseconditions. Transgenic plants were self-pollinated and seeds werecollected from all of them at the end of the experiments.

Example 4 Inoculation of Transgenic Plants

Seedlings of 5-7 leaf stage grown in the greenhouse were challenged witheither TSWV-BL or GRSV. Both viruses were multiplied in N. benthamiana,tested by ELISA for tospoviruses and by indirect ELISA with specificantibodies raised against each virus. After multiplication andimmunotesting individual leaves were kept at −20° C. for inoculationexperiments. A week in advance to inoculation of transgenic plantsnon-transgenic N. benthamiana was infected with the frozen leaves toserve as a source of fresh virus inoculum. In every round of inoculationof no more than 50 plants per set, 4 non-transgenic plants were includedas a control. Infected leaves were ground in a 0.033 M KH₂PO₄, 0.067 MK₂HPO₄, and 0.01 M Na₂SO₃ cold buffer. Ten-fold diluted leaf extract wasimmediately applied onto carborundum-dusted leaves, and subsequentlyrinsed with water. Symptoms were recorded daily starting 5 days afterinoculation, but only weekly 2 weeks after inoculation and until theplants set seeds. At least one leaf was taken from every plant beforeinfection and stored at −20° C. for further RNA extraction.

Example 5 Plant DNA and RNA Extraction

DNA from 100 mg of transgenic and non-transgenic N. benthamiana leaveswas extracted according to Fulton et al., “Microprep Protocol forExtraction of DNA from Tomato and Other Herbaceous Plants,” PlantMolecular Biology Reporter 13(3):207-209 (1995), which is herebyincorporated by reference in its entirety, from plants 3 weeks old. DNAwas kept in water at −20° C. until used for PCR or Southern analysis.RNA was extracted before and after infection from plants 3 and 6 weeksold, respectively, according to Napoli et al., “Introduction of aChimeric Chalcone Synthase Gene into Petunia Results in ReversibleCo-suppression of Homologous Genes in Trans,” Plant Cell 2:279-290(1990), which is hereby incorporated by reference in its entirety, using100 mg of leaves. In all cases, concentration was determined byspectrophotometry at 260 nm, and integrity in agarose gels for DNA anddenaturing agarose gels for RNA.

Example 6 Northern and Southern Analysis

Probes for hybridization by Southern and Northern analysis were labeledwith α-³²P-dATP by random priming according to Feinberg et al., “ATechnique for Radiolabeling DNA Restriction Endonuclease Fragments toHigh Specific Activity,” Anal. Biochem. 132(1):6-13 (1983), which ishereby incorporated by reference in its entirety. For Southern analysis15 μg of DNA from transgenic and non-transgenic lines were digested withan excess of restriction enzymes for at least 16 hours. Electrophoresis,transfer, and DNA fixation onto nylon membranes were performed asrecommended by the manufacturer (NEN Life Sciences, Boston, Mass.).Hybridization was carried out with the homologous ¾N gene fragment ofthe analyzed samples. After 24 hours of hybridization at 65° C.,membranes were washed according to manufacturer's recommendations (NENLife Sciences, Boston, Mass.). For Northern analysis, after 24 hourshybridization at 60° C., membranes were washed as before. After washingthe membrane was put in contact with an auto radiographic film fordifferent intervals of time. For Northern analysis membranes werestripped and hybridized with an α-³²P-dATP-labeled actin gene.

Example 7 Software

For the design, virtual assembly, and alignment of all synthetic andnative sequences the DNAStar program was used. Sequences were used toperform a BLAST search for homologous sequences at the GenBank database.For measuring band intensities both in photographed gels and in X-rayfilms the NIH Scio Image 1.59 software was used.

Example 8 Design and Synthesis of ¾N TSWV-BL N Gene.

Sequence modifications made to a DNA fragment are generally limited bythe availability of restriction sites, but also by the ability ofmutating at random, or even site-specifically, the gene under analysis.When trying to establish how important nucleotides changes are, both thenumber and the location of nucleotide changes must be assessed undercontrolled conditions. This is, by far, the most important requisite inthe design for making synthetic genes with specific desired properties,to test different hypothesis, and to attain specific plant phenotypes.Based on a sequence previously known to confer resistance, new fragmentsof 216 bp of TSWV N gene were designed with nucleotides changes torender scattered, 5′ end, middle and 3′ end region constructs differingby 5% and 15%, and scattered 10% and 20% from the native N TSWV-BL gene.A starting assumption was that in modifying the nucleotide sequence of aprevious fragment proven to confer resistance in transgenic plants, apoint would be reached in which resistance was no longer viable. In thisway, information could be gathered regarding what extent of similarityis important to trigger post-transcriptional gene silencing, and, evenmore importantly, on how the location of nucleotide changes affect thephenotype of transgenic plants when challenged with the homologousvirus.

Using the strategy originally devised by Stemmer et al., “Single-StepAssembly of a Gene and Entire Plasmid from Large Numbers ofOligodeoxyribonucleotides,” Gene 164(1):49-53 (1995), which is herebyincorporated by reference in its entirety, the constructs indicated byTable 4 were synthesized. The engineered N gene fragment of TSWV-BL (thelettuce isolate of TSWV) is the nucleotide sequence designated as ¾N byPang et al., “Nontarget DNA Sequences Reduce the Transgene LengthNecessary for RNA-Mediated Tospovirus Resistance in Transgenic Plants,”Proc. Natl. Acad. Sci. USA, 94:8261-8266 (1997), which is herebyincorporated by reference in its entirety), which corresponds to bases2668-2373 (antisense orientation) according to Pang et al., “Resistanceto Heterologous Isolates of Tomato Spotted Wilt Virus in TransgenicPlants Expressing its Nucleocapsid Protein Gene,” Phytopathology, 82:1223-29 (1992), which is hereby incorporated by reference in itsentirety. All fragments were 216 nt long, but adding the restrictionrecognition sites and random nucleotides introduced for cloningpurposes, all assembled products are 240 nt long. No cloning from nativevirus was required. Even the native version of the ¾N gene fragment wassynthetic, but also used as controls were ¾N-transgenic segregatinglines previously obtained in this laboratory. In this sense thesynthetic N gene fragment here used is identical to the one published byPang et al., “Resistance to Heterologous Isolates of Tomato Spotted WiltVirus in Transgenic Plants Expressing its Nucleocapsid Protein Gene,”Phytopathology 82: 1223-29 (1992), which is hereby incorporated byreference in its entirety, and all modified constructs derived from thissingle original sequence. This sequence was chosen as a baseline due toits short size and its ability to confer good level of resistance toTSWV when transcriptionally fused to GFP gene. The design of the oligosand the changes introduced to the N sequence were performed by computerusing the DNAStar software program. For every construct, all oligosrequired for their synthesis were individually designed and thecomplementary strand deduced using the DNAStar software. When all oligosequences were deduced they were introduced in a different program ofthe same software to allow its virtual assemblage. All of them, withoutexception, gave a unique contig that matches 100% with the intendedsequence when assembled as indicated in Table 4. That is, no otherassemblage product was produced in this simulation-like exercise.Afterwards, computer-simulated restriction analysis were performed toassess whether the introduced changes created BamHI, XhoI, KpnI, and/orHindIII restriction sites that would eventually complicate the cloningand subcloning process. All 10 newly-created sequences harbored only therestriction sites purposely introduced in the amplification oligos forcloning purposes (BamHI and XhoI). In all cases, the introducednucleotide changes were transversions; meaning that no overall change inG+C content is expected. The 10 constructs created are listed in Table4. TABLE 4 % Changes Location Assembly Primers Amplification PrimersNone TS-N-2H, TS-N-3H, TS-N-4H, TS-N-1AH, TS-N-1AC TS-N-5H, TS-N-6H,TS-N2C, TS-N-3C, TS-N-4C, TS-N-5C, TS-N-6C 5 Scattered TS-5-2H, TS-5-3H,TS-5-4H, TS-N-1AH, TS-5-1AC TS-5-5H, TS-5-6H, TS-5-2C, TS-5-3C, TS-5-4C,TS-5-5C, TS-5-6C 5 5′ TS-N-2H, TS-N-3H, TS-N-4H, TS-5-5′-1AH, TS-N-1ACTS-N-5H, TS-N-6H, TS-N2C, TS-N-3C, TS-N-4C, TS-N-5C, TS-5-5′-6C 5 MiddleTS-N-2H, TS-N-3H, TS-5-m-4H, TS-N-1AH, TS-N-1AC TS-N-5H, TS-N-6H,TS-N2C, TS-N-3C, TS-5-m-4C, TS-N-5C, TS-N-6C 5 3′ TS-N-2H, TS-N-3H,TS-N-4H, TS-N-1AH, TS-5-3′-1AC TS-N-5H, TS-5-3′-6H, TS-N2C, TS-N-3C,TS-N-4C, TS-N-5C, TS-N-6C 10 Scattered Rec2-2H, Rec2-3H, Rec2-4H,Rec2-1AH, Rec2-1AC Rec2-5H, Rec2-6H, Rec2-2C, Rec2-3C, Rec2-4C, Rec2-5C,Rec2-6C 15 Scattered TS-15-2H, TS-15-3H, TS-15-4H, TS-15-1AH, TS-15-1ACTS-15-5H, TS-15-6H, TS-15-2C, TS-15-3C, TS-15-4C, TS-15-5C, TS-15-6C 155′ TS-15-5′-2H, TS-N-3H, TS-N-4H, TS-10-5′-1AH, TS-N-5H, TS-N-6H,TS-N2C, TS-N-1AC TS-N-3C, TS-N-4C, TS-N-5C, TS-15-5′-6C 15 MiddleTS-N-2H, TS-N-3H, TS-15-m-4H, TS-N-1AH, TS-N-1AC TS-N-5H, TS-N-6H,TS-N2C, TS-15-m-3C, TS-10-m-4C, TS-N-5C, TS-N-6C 15 3′ TS-N-2H, TS-N-3H,TS-N-4H, TS-N-1AH, TS-N-5H, TS-15-3′-6H, TS-10-3′-1AC TS-15-3′-2C,TS-N-3C, TS-N-4C, TS-N-5C, TS-N-6C 20 Scattered TS-20-2H, TS-20-3H,TS-20-4H, TS-20-1AH, TS-20-1AC TS-20-5H, TS-20-6H, TS-20-2C, TS-20-3C,TS-20-4C, TS-20-5C, TS-20-6C

The strategy here used was devised by Stemmer et al., “Single-StepAssembly of a Gene and Entire Plasmid from Large Numbers ofOligodeoxyribonucleotides,” Gene 164(1):49-53 (1995), which is herebyincorporated by reference in its entirety, to assemble and amplify asynthetic bla (ampicillin resistance) gene in E. coli. Briefly, a set ofdifferent oligos corresponding to both strands of the DNA of interest issynthesized and mixed together in a PCR tube. After transformation andplasmid DNA extraction, samples were digested and those that yielded theexpected size fragment were sequenced. In all cases, the sequence of thecloned gene matched perfectly with the virtual version of the designedfragment obtained by computer analysis. One example is shown in FIG. 3.All of the different synthetic constructs have the exact sequencegenerated by computer analysis.

Example 9 Both Degree of Similarity and Location of Nucleotide ChangesAffect the Ability of a ¾N Transgene in Conferring Resistance to TSWV-BL

The first goal was to determine how location of nucleotide changes, andthe amount of those changes, affect the ability of transgenic plants tobe resistant to TSWV. The fragment chosen for engineering (i.e., thethird fourth of the TSWV N gene, ¾ N) proved to be very effective whentranscriptionally fused to GFP. The nucleotide sequence of such afragment was modified and used to transform N. benthamiana plants.Plants were analyzed by PCR for nptII and ¾ N gene; as shown in FIG. 4,and some selected lines for every construct were tested by Southern andNorthern analysis. Results of inoculation are shown in Table 5, andexamples of susceptible and resistant plants in FIG. 5. TABLE 5 Responseof N. benthamiana Transgenic Plants to TSWV Infection: Summary ofResults by Construct Transgenic lines/ Inserted transgene^(a)phenotype^(b) % Changes Position Total Susceptible Delay Resistant  5%5′ 57 23 2 32 (56.1%) M 38 15 — 23 (60.5%) 3′ 51 30 — 21 (42.2%)Scattered 33 13 — 20 (57.5%) 15% 5′ 64 31 — 33 (51.5%) M 61 26 6 29(47.5%) 3′ 36 20 — 16 (44.4%) Scattered 20 14 1  5 (25.0%) 20% Scattered50 43 5 2 (4.0%) None Synthetic N 36 14 — 22 (61.1%) Native N^(c) 50 216 23 (46.1%) Native N^(d) 19 5 3 11 (57.8%)^(a)All genes, native or synthetic, correspond to the third fourth ofthe N gene of TSWV-BL.^(b)In vitro or seed-derived plants were kept in the greenhouse andinoculated at the 5-7 leaves stage with a 1:10 dilution of the virus,and scored daily until they set flowers.^(c)Segregating R1 plants from Pang et al., “Nontarget DNA SequencesReduce the Transgene Length Necessary for RNA-Mediated TospovirusResistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997)which is hereby incorporated by reference in its entirety)N-transgenic line 5 seeds.^(d)Segregating R1 plants from Pang et al., “Nontarget DNA SequencesReduce the Transgene Length Necessary for RNA-Mediated TospovirusResistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997) which is hereby incorporated by reference in itsentirety) N-transgenic line 22 seeds.

As expected, a higher number of nucleotide changes that diminishsequence similarity between the transgene and the incoming virus have adetrimental effect on viral resistance. For 5%, 15%, and 20% changes,the number of resistant transgenic plants were 57.5%, 25%, and 2%,respectively. The highest number of resistant plants was obtained withthe synthetic, unmodified version of ¾N gene (61.1%). Even at levels ofhomology as low as 80%, it is still possible to obtain some level ofresistance. The 2 out of 50 plants with 20% changes that showed somelevel of resistance are under analysis in the second generation. Thistrend is also evident when all plants with a similar amount of changesare added up, regardless of the location, as shown in Table 6. TABLE 6Response of N. benthamiana Transgenic Plants to TSWV Infection (Summaryof results by percentage of change)* Inserted transgene^(a) Transgeniclines/phenotype^(b) % Changes Number Susceptible Delay Resistant  5 17981 2 96 (53.6%) 15 181 91 7 83 (45.9%) 20 50 43 5 2 (4.0%) None:synthetic 47 19 — 28 (59.6%) None: native^(c) 50 21 6 23 (46.0%) None:native^(d) 19 5 3 11 (57.8%)*Sum of all positions for same amount of changes, according to Table 3.^(a)All genes, native or synthetic, correspond to the third fourth ofthe N gene of TSWV-BL^(b)In vitro or seed-derived plants were kept in the greenhouse andinoculated at the 5-7 leaves stage with a 1:10 dilution of the virus,and scored daily until they set flowers.^(c)Segregating R1 plants from Pang et al., “Nontarget DNA SequencesReduce the Transgene Length Necessary for RNA-Mediated TospovirusResistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997) which is hereby incorporated by reference in itsentirety) N-transgenic line 5 seeds.^(d)Segregating R1 plants from Pang et al., “Nontarget DNA SequencesReduce the Transgene Length Necessary for RNA-Mediated TospovirusResistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA, 94:8261-66 (1997) which is hereby incorporated by reference in itsentirety) N-transgenic line 22 seeds.

It also appears that location of nucleotide changes in the transgene canexert an impact on transgenic resistance. Both for sequences with 5% and15% changes, the number of resistant plants with engineered changes atthe 3′ end was lower when compared with plants with changes clustered atthe 5′ end or the middle section of the gene fragment. Apparently, the3′ end is somehow more critical in conferring resistance than the 5′end, regardless of the length of the modified 3′ end, as shown in Table5 and in Table 7, which shows a summary of results by location ofchange. TABLE 7 Response of N. benthamiana Transgenic Plants to TSWVInfection Inserted transgene^(a) Transgenic lines/phenotype^(b) Locationof change Number Susceptible Delay Resistant 5′ end 121 54 2 65 (53.8%)3′ end 87 50 — 37 (42.3%) middle 99 41 6 52 (52.5%) None: synthetic 4719 — 28 (59.6%) None: native^(c) 50 21 6 23 (46.0%) None: native^(d) 195 3 11 (57.8%)*Sum of all different percentage of changes for same position, accordingto Table 3.^(a)All genes, native or synthetic, correspond to the third fourth ofthe N gene of TSWV-BL.^(b)In vitro or seed-derived plants were kept in the greenhouse andinoculated at the 5-7 leaves stage with a 1:10 dilution of the virus,and scored daily until they set flowers.^(c)Segregating R1 plants from Pang et al., “Nontarget DNA SequencesReduce the Transgene Length Necessary for RNA-Mediated TospovirusResistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA. 94:8261-66 (1997) which is hereby incorporated by reference in itsentirety) N-transgenic line 5 seeds.^(d)Segregating R1 plants from Pang et al., “Nontarget DNA SequencesReduce the Transgene Length Necessary for RNA-Mediated TospovirusResistance in Transgenic Plants,” Proc. Natl. Acad. Sci. USA. 94:8261-66 (1997) which is hereby incorporated by reference in itsentirety) N-transgenic line 22 seed.

Example 10 A Synthetic ¾N Gene, 216 bp Long Can Confer Resistance toBoth TSWV and GRSV

Transgenic plants transformed with ¾N gene fused to GFP can conferresistance to TSWV but not to the related tospovirus Groundnut RingspotVirus (GRSV). TSWV and GRSV are 78% similar, and it is possible that thelack of resistance of transgenic plants challenged with GRSV is due to alevel of homology which is insufficient to target the GRSV genome fordegradation, even if post-transcriptional gene silencing is triggered.To further explore the possibility that modifying the TSWV ¾N transgenewould allow broader viral resistance of transgenic plants, a gene wassynthesized that is, in toto, approximately 90% similar to thecorresponding fragments of both TSWV and GRSV. To design this sequence,a ¾N TSWV gene was modified to make it more homologous to the GRSV Ngene nucleotide sequence. In doing so, of course, the similarity to thenative TSWV N gene decreased, the net result being that the artificialsequence presented here is 90% similar to both TSWV N gene and itscounterpart in GRSV. The nt 1417-1632 (in sense form) of TSWV and nt560-775 of GRSV were used. The newly created synthetic sequence (calledRec2 herein), when compared with its parental ¾N TSWV-BL gene sequence,has the following changes: one insertion and one deletion, plus 22 basechanges (ca. 10% changes compared with the native sequence of ¾N TSWVgene). Similarity of TSWV N gene dropped from 100% to 90% according tosequence alignment. Similarity to GRSV rose from 73% to 89% according tothe same analysis. The homologous gene of Tomato Chlorotic Spot Virus(TCSV), another member of the same group as TSWV and GRSV, is also 89%similar to Rec2, as seen in Table 8. In FIG. 6, all modifications arehighlighted and uninterrupted stretches of full similarity are alsoindicated. TABLE 8 Sequence Similarity (%) Among 216 bp ¾ N GeneFragments of Selected Tospoviruses and Recombinant, Synthetic SequenceRec2. TSWV TCSV GRSV Rec2 TSWV 100.0 78.7 73.1 90.0 TCSV 100.0 77.3 89.0GRSV 100.0 89.0 Rec2 100.0The values here tabulated were originated by annealing the sequences bypairs instead of a single, multiple analyses. For that purpose BLAST 2Sequences algorithm and program was used.

Approximately 27% of the transgenic lines analyzed showed a good levelof resistance against either TSWV or GRSV. At first glance, plantstransformed with constructs differing by 10% from its native sourcesupport the trend observed for scattered changes. The proportion ofresistant plants with engineered constructs differing by 10% liesbetween the values observed for plants transformed with 5% changes(56.1%) and 15% changes (25%)—although very similar for the latter. Itmust be taken into consideration that changes for construct Rec2 werenot made at random, and the distance between changed nucleotides is noteven; this was the case for all the other scattered changes constructs.A second important observation is that the original hypothesis—i.e.,that a DNA fragment can be engineered that confers resistance to twodifferent viruses whose “natural” similarity is below a threshold totrigger post-transcriptional gene silencing—can be effectively appliedto obtain transgenic plants with multiple viral resistance. Using thecurrent model for post-transcriptional gene silencing andRNA-interference, it would be possible to engineer a construct not onlyto confer resistance to three-distantly related virus, but also tomanipulate other genetic traits using a single, short, gene fragmentbuilt from the information of different gene systems.

Example 11 Response to Infection of Transgenic Nicotiana BenthamianaPlants

Nicotiana benthamiana plants were transformed with a ¾ N TSWV-BL genefragment modified for a 90% similarity to TSWV, GRSV and TCSV accordingto the examples above. The transgenic plants were then challenged withTSWV-BL or GRSV. Table 9 shows the results of virus resistance for thetransgenic plants. TABLE 9 Response to Infection of Transgenics With a ¾N TSWV-BL Modified Fragment Challenging virus Total Plants ResistantSusceptible TSWV-BL 10 3 (30%) 7 GRSV 15 4 (27%) 11 Total 25 7 (28%) 18

Example 12 A Synthetic Sequence Derived From Three PRSV Isolates

A set of synthetic genes were designed according to the presentinvention to target for degradation the RNAs of three different isolatesof PRSV. The isolates were the Keaau (Hawaii) strain (“KE”), theThailand strain (“TH”), and the severe Taiwan strain (“YK”). Thestarting sequences were the conserved and variable regions, respectivelyof the coat protein (“CP”) gene. For the analysis of selected DNAsequences, the DNAStar package (LaserGene Software, Madison, Wis.) wasused for editing and aligning. DNAStar and the Blast 2 Sequences fromNCBI were used to compare sequences by pairs. To compare the sequence ofinterest with the GenBank database, the Blast program was used (Altschulet al., “Gapped BLAST and PSI-BLAST: A New Generation of ProteinDatabase Search Programs, Nucleic Acids Research 35:3389-3402 (1997),which is hereby incorporated by reference in its entirety). For multiplealignment both ClustalW and Map were employed (both of them freelyavailable), whereas for searching motifs MEME and MAST yielded the bestresults (Bailey et al, “New Tools for Quantifying Molecular Diversity,”Pharmainformatics 6-7 (1999); Bailey et al., “Combining Evidence Usingp-Values: Application to Sequence Homology Searches,” Bioinformatics14:48-54 (1998), which are hereby incorporated by reference in theirentirety). DNAStar can also be used to generate phylogenetic trees andsequence similarity matrices. The CP gene sequences employed for thiswork were isolated and were sequenced in inventors' laboratory.

Alignments were also analyzed with the GeneDoc software Nicholas et al.,“GeneDoc: A Tool For Editing and Annotating Multiple SequenceAlignments” Software Ver. 2.6.002 (1997), and similarity trees generatedwith the Tree View software (Page, Software version 1.6. (2001), both ofthem freely available on the web.

The similarity matrix for the conserved and variable regions of the CPgene for the KE, TH and YK isolates and the synthetic sequence createdfor the conserved region and the variable region is shown below in Table10. TABLE 10 Conserved region (203 nt) Variable region (209 nt) KE TH YKSyncon KE TH YK Synvar KE 100.0 94.1 94.1 97.0 KE 100.0 83.5 83.7 91.9TH 100.0 96.1 97.0 TH 100.0 85.4 91.7 YK 100.0 97.0 YK 100.0 91.9 Syncon100.0 Synvar 100.0Isolates under comparison are KE, Keaau (Hawaii); TH, Thailand; YK,Severe Taiwan strain.“Syncon” stands for the synthetic sequence for the conserved region, and“Synvar” for variable region.

FIG. 7 shows the starting sequences for the conserved regions of the CPgene of TH (SEQ ID NO: 6), KE (SEQ ID NO: 7) and YK (SEQ ID NO: 8)compared to each other and to the sequence of the modified syntheticnucleic acid (SEQ ID NO: 5) sequence generated for targeting these PRSVisolates. The underlined portions identify segments of more than 20 ntlong of perfect similarity. The nucleotide changes differing from thesynthetic sequence are shown in lowercase. Because the homology of thethree starting sequences chosen for the isolates was high, a highhomology (97%) synthetic sequence with a low variation (range: 0%) wascreated, as shown in Table 10.

FIG. 8 shows the distribution of dissimilar nucleotides compared to thesynthetic gene (SEQ ID NO: 9) for the variable regions of the of the CPgene of TH (SEQ ID NO: 10) KE (SEQ ID NO: 11) and YK (SEQ ID NO: 12).The underlined portions identify segments of more than 20 nt long ofperfect similarity. The nucleotide changes differing from the syntheticsequence are shown in lowercase. The starting homologies among thevariable regions of the CP gene are shown in Table 10. In this case, thehomology of the starting sequence was 84.2%, and was raised to 91.8%when compared to the synthetic sequence. In addition, the variationamong the starting sequences relative to the synthetic is very low, >1%.

Note that in both cases, as seen in FIG. 7 and FIG. 8, the longfragments of perfect similarity are present in all of the three isolateswhen compared with the sequence of the synthetic transgene. Therationale of this approach is based on the proposed concept that themediator molecules of dsRNA that trigger gene silencing seem to be 20-27nt long (Hamilton et al., “A Species of Small Antisense RNA inPosttranscriptional Gene Silencing in Plants,” Science 286:950-952(1999); Waterhouse et al., “Role of Short RNAs in Gene Silencing,”Trends in Plant Science 6:297-301 (2001), which are hereby incorporatedby reference in their entirety). This is not meant to limit in any waythe size of similar segments chosen for a synthetic sequence of thepresent invention in comparison with the desired trait DAN molecules.

Example 13 A Synthetic Sequence Derived From Diverse Potyviruses

A synthetic sequence was generated according to the present invention totarget for degradation a variety of different potyviruses. The desiredtrait is potyvirus resistance in different crop plants, both dicots andmonocots. Therefore, the complete potyvirus genome was the startingpoint for this synthetic sequence. Nineteen sequences from potyviruseswhose complete genomes have been already sequenced, having an overallhomology of 34-83%, were chosen as starting nucleotide sequences forcomparative analysis. The nucleotide sequences were obtained from theGenBank database. A variety of genes of the potyvirus genome werepotential candidates for applying the method of the present invention.Table 11, below, gives the numerical coordinates of all the genes of thepotyviruses used for this analysis.

Table 12 shows an analysis of percent of nucleotide identity for alldifferent genes in selected potyviruses. Using this type of analysesassists in selecting a gene within a genome suitable for generating thesynthetic nucleic acid molecule of the present invention. TABLE 11 HC-NIa- NIa- VIRUS 5′UTR^(a) P1(1)^(b) PRO^(c) P3^(d) 6K1^(e) CI^(f)6k2^(g) VPg^(h) Pro^(i) NIb^(j) CP(2)^(k) 3′UTR^(l) Bean common mosaic 1131 1400 2771 3812 3968 5870 6029 6599 7328 8876 9737 Japanese yammosaic 1 154 1135 2509 3574 3730 5662 5821 6397 7126 8680 9553 Maizedwarf mosaic 1 140 839 2219 3260 3461 5375 5534 6101 6827 8390 9263Papaya ringspot 1 86 1727 3098 4133 4289 6210 6365 6932 7646 9197 10199Peanut Mottle 1 123 1086 2460 3507 3663 5565 5724 6294 7032 8586 9423Peanut Stripe 1 134 1463 2834 3875 4031 5933 6092 6662 7391 8939 9800Pea seed-borne 1 144 1338 2715 3771 3927 5835 5994 6567 7305 8865 9738Pepper mottle 1 168 1029 2397 3480 3636 5538 5694 6258 6996 8553 9372Plum pox 1 147 1071 2445 3495 3651 5556 5715 6294 7023 8577 9522 PotatoY 1 185 1056 2427 3468 3624 5529 5688 6255 7010 8573 9373 Potato A 1 1621037 2405 3500 3656 5558 5714 6278 6984 8532 9339 Ryegrass mosaic 1 113881 2246 3302 3461 5372 5531 6110 6833 8153 9371 Scallion mosaic 1 110743 2114 3176 3332 5264 5423 5999 6728 8279 9113 Soybean mosaic N 1 1321056 2427 3468 3624 5526 5685 6255 6984 8535 9330 Sugarcane mosaic 1 150849 2229 3260 3471 5385 5544 6111 6837 8400 9339 Sweet Potato feathery 1118 2110 3484 4540 4696 6625 6784 7360 8089 9652 10597 mottle Tobaccoetch 1 144 1056 2433 3474 3633 5532 5691 6255 6981 8517 9306 Turnipmosaic 1 131 1217 2591 3656 3812 5744 5903 6479 7208 8759 9623 Zucchiniyellow mosaic 1 139 1069 2437 3475 3631 5533 5692 6262 6991 8542 9379(1) = Including the ATG codon;(2) = Not including the Stop codon;^(a)= 5′ untranslated region;^(b)= Protein 1;^(c)= Helper component Protease;^(d)= protein 3;^(e)= 6 kda ‘protein 1’;^(f)= cytoplasmic inclusion protein;^(g)= 6 kda ‘protein 2’;^(h)= Nuclear inclusion protein a-viral genome linked protein;^(i)= protease nuclear inclusion protein a;^(j)= Nuclear inclusion protein b;^(k)= Coat Protein;^(l)= 3′ untranslated region.

TABLE 12 Percent Maximum Similarity Workable Length (%) Length Gene (nt)ClustalW (nt) Quality 5′ UTR  85-184 22-80 30 Low P1  633-1992 19-85 20Low HC-Pro 1368-1380 39-86 1: 85 Medium → 2: 158 Medium 3: 90 Medium P31035-1083 30-81 83 Low 6K1 156-211 27-86 59 Low CI 1899-1932 50-82 1:152 Medium 2: 46 High → 3: 188 Medium 6K2  96-159 27-81 <20 Low NIa Pro706-738 45-88 1: 94 Medium 2: 190 Low NIa VPg 564-579 42-87 → 1: 121Medium 2: 105 Medium NIb 1320-1563 54-86 → 1: 211 High → 2: 273 High CP 801-1218 37-91 → 1: 201 High → 2: 250 Medium 3′ UTR 165-331 18-93 NoneNone Whole  9324-10820 34-83 Indicated Indicated genome

From these 19 potyviruses, the group was narrowed down to the 9potyviruses, shown along with their accession numbers, in Table 13,below. TABLE 13 Accession Plant Species Gene Number Bean common mosaicvirus Complete Genome NC 003397 Maize dwarf mosaic virus Complete GenomeNC 003377 Peanut mottle virus Complete Genome NC 002600 Pea seed-bornemosaic virus Complete Genome AJ252242 Pepper mottle virus CompleteGenome NC 001517 Potato virus Y Complete Genome NC 001616 Soybean mosaicvirus N Complete Genome NC 002634 Sugarcane mosaic virus Complete GenomeNC 003398 Zucchini yellow mosaic virus Complete Genome NC 003224

The nine selected potyvirus genes were compared and analyzed usingDNAStar package (LaserGene Software, Madison, Wis.) which was utilizedfor editing and aligning. DNAStar and the Blast 2 Sequences from NCBIwere used to compare sequences by pairs. To compare the sequence ofinterest with the GenBank database, the Blast program was used (Altschulet al., “Gapped BLAST and PSI-BLAST: A New Generation of ProteinDatabase Search Programs, Nucleic Acids Research 35:3389-3402 (1997),which is hereby incorporated by reference in its entirety). For multiplealignment both ClustalW and Map were employed (both of them freelyavailable), whereas for searching motifs MEME and MAST yielded the bestresults (Bailey et al, “New Tools for Quantifying Molecular Diversity,”Pharmainformatics 6-7 (1999); Bailey et al., “Combining Evidence Usingp-Values: Application to Sequence Homology Searches,” Bioinformatics14:48-54 (1998), which are hereby incorporated by reference in theirentirety). FIG. 9 shows the CLUSTALW alignment of the nine selectedpotyvirus sequences and their consensus sequence. The similarity amongthe nine native sequences, having approximately 200 nucleotides, wasdetermined to be 64.4-92.1% (range: 27.7%). The similarity with theconsensus sequence identified for the nineteen sequences was found tobe: 75.2-86.6% (range: 11.4%). Using Formula I supra, it was determinedthe total number of changes would be approximately 20. A referencesequence was chosen from among the starting sequences, and nucleotidechanges were made by reviewing the alignment data of FIG. 9 anddetermining those modifications that would result in a single syntheticsequence with a homology of greater than 80, but less than 100% relativeto each starting sequence, and which would also result in variation inpercent identity narrower than 3%. After approximately 20 nucleotideswere changed, the CLUSTALW alignment was run again as shown in FIG. 10.The similarity with the synthetic gene was determined to be 74.3-78.2%(range: 3.9%). Thus, a single 202 nt synthetic gene was designed in thisstep-wise fashion. This synthetic modified potyvirus nucleic acidmolecule has a nucleotide sequence of SEQ ID NO: 23 as follows:atgggagttg tgatgaatgg cctcatggtt tggtgcattg 60 aaaatggaac ctcaccaaacatcaatggag tatgggctat gatggatggg gacgaacaag 120 ttgagtttcc attaaagccagtgattgaga atgcaaagcc aacttttcga caaatcatgc 180 atcatttttc agatgcagcagaagcttaca tagagtagag ca 202

This example illustrates that the present invention can be applied to ahighly divergent trait DNA group with significant results in terms ofcreating a molecule that is a marked improvement over the variation inhomology found among the members of the group in nature. The range ofhomology was reduced from 27.7% for the starting sequences themselves to3.9% for the starting sequences in comparison to the modified sequence.It is highly probable that with further manipulations in accordance withthe present invention, a suitable single modified nucleic acid moleculewill be created that has yet a lower range in homology with respect tothe starting trait DNA group.

Note that more than one “block” or region of homology can be used fromdifferent genes, and sequences can be grouped separately to createchimeras of the same block or chimeras of different blocks. The onepresented here is just one example of a multitude of possibilities.

Example 14 A Synthetic Sequence To Delay Fruit Ripening Using ASynthetic Polygalacturonase Gene Derived From Different Plants

A synthetic sequence was generated according to the present invention totarget for degradation of different genes of the same gene family in thesame plant, in this case the polygalacturonase (“PG”) gene of tomato.The desired trait to be imparted by this synthetic gene is delayed fruitripening.

PG is a well-characterized enzyme family implicated in the disassemblyof pectin that accompanies many stages of plant development, inparticular, fruit ripening (Sheehy et al., “Reduction ofPolygalacturonase Activity in Tomato Fruit by Antisense RNA,” Proc.Natl. Acad. Sci. USA 85:8805-8809 (1988); Smith et al., “Antisense RNAInhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes,”Nature 334:724-726 (1988); Giovannoni et al., “Expression of a ChimericPolygalacturonase Gene in Transgenic Rin (Ripening Inhibitor) TomatoFruit Results in Polyuronide Degradation But Not Fruit Softening,” PlantCell 1:53-63 (1989), which are hereby incorporated by reference in theirentirety). Delivery to market of mature, full-flavored, andunadulterated fruits, such as tomato and papaya, is a challenge toproducers, especially when long-distance transport is involved.Therefore, the ability to control fruit ripening is a commerciallydesirable trait.

Five reported sequences for the polygalacturonase gene in Lycopersiconesculentum were obtained from GenBank, as shown Table 14. TABLE 14GenBank Accession Plant Species Gene Number LycopersiconPolygalacturonase 3 (TAPG3) gene AF000999 esculentum LycopersiconPolygalacturonase 5 (TAPG5) gene AF001003 esculentum LycopersiconPolygalacturonase 4 (TAPG4) gene AF001002 esculentum LycopersiconPolygalacturonase 2 (TAPG2) gene AF001001 esculentum LycopersiconPolygalacturonase 1 (TAPG1) gene AF00100 esculentum

Once the sequence were obtained, DNAStar package (LaserGene Software,Madison, Wis.) was used for editing and aligning. DNAStar and the Blast2 Sequences from NCBI were used to compare sequences by pairs. Tocompare the sequence of interest with the GenBank database the Blastprogram was used (Altschul et al., “Gapped BLAST and PSI-BLAST: A NewGeneration of Protein Database Search Programs, Nucleic Acids Research35:3389-3402 (1997), which is hereby incorporated by reference in itsentirety). For multiple alignment both ClustalW and Map were employed(both of them freely available), whereas for searching motifs MEME andMAST yielded the best results (Bailey et al, “New Tools for QuantifyingMolecular Diversity,” Pharmainformatics 6-7 (1999); Bailey et al.,“Combining Evidence Using p-Values: Application t Sequence HomologySearches,” Bioinformatics 14:48-54 (1998), which are hereby incorporatedby reference in their entirety). FIG. 11 shows the alignment of the fiveselected tomato PG sequences and their consensus sequence. Thesimilarity among the five native sequences was determined to be86.0-96.1% (range: 10.1%). The similarity with the consensus sequencewas determined to be 90.3-96.6% (range: 6.3%). Using Formula I supra, itwas determined that approximately 20 nucleotides could be expected to bemodified in order to create a suitable synthetic sequence to target PGRNA. Using a reference sequence chosen from among the starting PGsequences, nucleotides were changed step-wise to create a single 207 ntsynthetic gene. The similarity with the synthetic gene was determined tobe 92.3-93.2% (range: 0.9%) for all of the starting tomato genes. Themodified nucleic acid molecule has a high percent homology, and,importantly, also has an decreased variation in similarity, as comparedto all the starting sequences. This means that the synthetic nucleicacid molecule is more likely to be an effector of RNA degradation in avariety of plants because it is more similar, on average, than many ofthe native starting sequences were to one another, or to the consensussequence. FIG. 12 shows the percent identity determined for the fivestarting sequences compared to the synthetic sequence. This syntheticgene is then cloned into an appropriate vector, as described aboveherein, and used to transform a tomato plant, plant cell, or plant line.The transgene can be engineered by creating a sequence having invertedrepeats that lead to the production of a dsRNA upon transcription in thetransgenic plant. Without being bound to a theory, a transgeneengineered in this way should lead to gene silencing of the endogenousgene, and, in this case, delayed plant ripening in the transgenic tomatoplant.

This synthetic modified tomato PG nucleic acid molecule has a nucleotidesequence of SEQ ID NO: 30 as follows: caaggagtga aggtgtcagc tccaggaaatagccccaata 60 ctgatggaat tcatgtacaa tcatcatctg gagttagtat tatgaaatcaaatattggta 120 ctggagacga ttgtatatct attggccctg gaacttcgaa cttatggattgaaggcattg 180 cttgtggccc tggccatgga ataagcattg gaagcttagg ctgggaa 207

Example 15 A Synthetic Gene Using Chalcone Synthase Genes in Petunia

A synthetic sequence was generated according to the present invention totarget for degradation different genes of the same gene family in thesame plant, using the chalcone synthase gene of Petunia. In flowers, aswell as food crops, the ability to manipulate color through geneticengineering is desirable because it is more predictable and lesstime-consuming than classic breeding methods. Thus, the desired trait inthis example is the species-specific inactivation of color developmentin Petunia. Petunia was also chosen as a representative of theapplication of the present invention to a dicot. Six reported sequencesfor the chalcone synthase gene of Petunia, shown below in Table 15 alongwith their GenBank accession numbers, were chosen. TABLE 15 GenBankAccession Plant Species Gene Number Petunia x hybrida chalcone synthase(chs) mRNA AF233638 Petunia hybrida chsJ gene for chalcone synthaseX14597 Petunia hybrida chsG gene for chalcone synthase X14595 Petuniahybrida chsF gene for chalcone synthase X14594 Petunia hybrida chsD genefor chalcone synthase X14593 Petunia hybrida chsB gene for chalconesynthase X14592

The genes of the six Petunia CHS genes were compared and analyzed usingboth ClustalW and MAP for multiple alignment. DNAStar package (LaserGeneSoftware, Madison, Wis.) was used for editing and aligning, and DNAStarand the Blast 2 Sequences from NCBI were used to compare sequences bypairs. To compare the sequence of interest with the GenBank database theBlast program was used (Altschul et al., “Gapped BLAST and PSI-BLAST: ANew Generation of Protein Database Search Programs, Nucleic AcidsResearch 35:3389-3402 (1997), which is hereby incorporated by referencein its entirety). FIG. 13 shows the homology of the starting sequencesand their consensus sequence. The similarity among the six sequences wasdetermined to be 76.1-86.6% (range: 10.5%). The similarity with theconsensus sequence was determined to be 83.6-93.3% (range: 9.7%). Asingle 134 nt synthetic gene, SEQ ID NO: 38, shown below, was manuallydesigned using the region of high homology within the identifiedconsensus sequences. The similarity with the synthetic gene wasdetermined to be 85.8-86.6% (range: 0.8%) for all of the startingPetunia genes. The synthetic modified nucleic acid molecule for PetuniaCHS not only has an improved degree of homology, having raised the leasthomologous starting sequence from 76.1% to 85.8%, but it also has anarrow range of variation in similarity compared to all startingsequences, i.e., 0.8% compared to a range of 10.5% variation insimilarity among the native starting sequences. This combination ofhomology greater than 80% and a narrow range of variation make thePetunia synthetic nucleic acid molecule a good candidate for degradingRNA in a target host, because the transcribed dsRNA is less likely to beperceived as foreign by its host, and will, therefore, be able toinitiate RNA degradation of the target host RNA and impart its intendedtrait. FIG. 14 shows the percent identity determined for the sixstarting sequences compared to the synthetic sequence. This syntheticgene is then cloned into an appropriate vector, using methods describedabove, and then used to transform a Petunia plant, plant cell or plantline. The transgene can be engineered by creating a sequence havinginverted repeats that lead to the production of a dsRNA upontranscription in the transgenic plant. Without being bound to a theory,a transgene engineered in this way should lead to gene silencing of theendogenous gene, and, in this case, disruption of color development inthe transgenic Petunia plant.

This synthetic modified Petunia CHS nucleic acid molecule has anucleotide sequence of SEQ ID NO: 38 as follows: aagctccttg gacttagtccatcagtcaag cgactaatga 60 tgtaccaaca aggttgcttt gctggtggca ctgtgcttcgattggcaaag gacttggctg 120 agaataacaa aggcgctcga gtccttgttg tgtg 134

Example 16 A Synthetic Gene Using Chalcone Synthase Genes in Sorghum

A synthetic sequence was generated according to the present invention totarget for degradation different genes of the same gene family in thesame plant, this time using the chalcone synthase gene of Sorghum.Sorghum was selected, because it is a grain crop of commercial value andis a representative of the application of the present invention to amonocot. Seven reported sequences for the chalcone synthase gene inSorghum, shown below in Table 16 along with their GenBank accessionnumbers, were chosen as starting sequences for analysis. TABLE 16GenBank Accession Plant Species Gene Number Sorghum bicolor chalconesynthase 7 (CHS7) gene AF152554 Sorghum bicolor chalcone synthase 6(CHS6) gene AF152553 Sorghum bicolor chalcone synthase 5 (CHS5) geneAF152552 Sorghum bicolor chalcone synthase 4 (CHS4) gene AF152551Sorghum bicolor chalcone synthase 3 (CHS3) gene AF152550 Sorghum bicolorchalcone synthase 2 (CHS2) gene AF152549 Sorghum bicolor chalconesynthase 1 (CHS1) gene AF152548

The genes of the seven Sorghum CHS genes were compared and analyzedusing both ClustalW and MAP for multiple alignment. DNAStar package(LaserGene Software, Madison, Wis.) was used for editing and aligning,and DNAStar and the Blast 2 Sequences from NCBI were used to comparesequences by pairs. To compare the sequence of interest with the GenBankdatabase the Blast program was used (Altschul et al., “Gapped BLAST andPSI-BLAST: A New Generation of Protein Database Search Programs, NucleicAcids Research 35:3389-3402 (1997), which is hereby incorporated byreference in its entirety). The alignment of the selected sorghumsequences are shown in FIG. 15. The similarity among the seven sequenceswas determined to be 95.9-97.9% (range: 2.0%). The similarity with theconsensus sequence was determined to be 93.5-99% (range: 5.5%). UsingFormula I supra, it was determined the total number of nucleotidechanges would be approximately 3. A reference sequence was chosen fromamong the starting sequences, and nucleotide changes were made byreviewing the alignment data of FIG. 15 and determining thosemodifications that would result in a single synthetic sequence with ahomology of greater than 80, but less than 100% relative to eachstarting sequence, and which would also result in variation in percentidentity of 3% or less. After the nucleotide changes were made thealignment was run again as shown in FIG. 16. The similarity with thesynthetic gene was determined to be 91.8-93.1% (range: 1.3%) for all ofthe starting Sorghum genes. This synthetic gene could then cloned intoan appropriate vector, using methods described above, and then used totransform a Sorghum plant, plant cell or plant line. The transgene canbe engineered by creating a sequence having inverted repeats that leadto the production of a dsRNA upon transcription in the transgenic plant.Without being bound to a theory, a transgene engineered in this wayshould lead to gene silencing of the endogenous gene, and, in this case,disruption of color development in the transgenic Sorghum plant. Thesynthetic modified Sorghum CHS nucleic acid molecule has a nucleotidesequence of SEQ ID NO: 47 as follows: cctcaaggag aagttcaaga ggatatgcgacaagtcgaag 60 atcaggaagc gttacatgca cttgacggag gagaacctag cggagaaccccaacatatgc 120 gcgtacaggg cgccgtcgct ggacgcccgc caggacatcg tggtggtggagatacccaag 180 ctaggcgagg ccgcggcgca gaaggcgatc aaagagtggg ggcagccgaattccaagatc 240 acgcacctcg tcttctgcac cacctccggc gtcgacatgc ctggcgccgactaccagctc 291 atcaagatgc t

Example 17 A Synthetic Sequence to Target the Same Gene Present inDifferent Plant Species (Same Trait, Different Species) UsingACC-Oxidase Gene in Different Plant Species

A synthetic sequence was generated according to the present invention totarget for degradation the same gene present in several different plantspecies. The desired trait is fruit ripening delay in different plantspecies. Sequences for 20 different genes of the ACC-oxidase geneinvolved in fruit ripening were used (see Table 17). From differentpossible ‘families’, a branch was selected that includes a varied groupof plants—some of them of economic value. TABLE 17 GenBank AccessionPlant Species Gene Number Actinidia deliciosa ACC oxidase homologueprotein mRNA M97961 Arabidopsis thaliana ACC oxidase (ACO2) mRNAAF016100 Arabidopsis thaliana At1g12010/F12F1_12 mRNA AY052694 Brassicajuncea 1-aminocyclopropane-1-carboxylate AF252628 oxidase (EFEMR2) mRNABrassica napus amino-cyclopropane-carboxylic acid L27664 oxidase exons1-4 Brassica oleracea mRNA for ACC oxidase (ACC0x1) X81628 Brassicaoleracea mRNA for ACC oxidase (ACC0x2) X81629 Brassica rapa subsp. mRNAfor ACC oxidase (acoii gene) AJ309322 rapa Brassica rapa subsp. mRNA forACC oxidase (acoi gene) AJ309321 rapa Carica papaya ripening-induced ACCoxidase mRNA AY077461 Carica papaya ACC oxidase gene AF320071Lycopersicon LE-ACO4 mRNA for 1- AB013101 esculentumaminocyclopropane-1-carboxylate oxidase Malus domestica ACC oxidase geneX89627 Malus domestica 1-aminocyclopropane-1-carboxylate AF015787oxidase (ACO2) gene Nicotiana glutinosa ACC oxidase (NGACO3) mRNA U62764Nicotiana glutinosa 1-aminocyclopropane-1-carboxylic acid U54566 oxidase(NGACO2) mRNA Pisum sativum1- aminocyclopropane-1-carboxylate oxidasemRNA M98357 Pyrus pyrifolia mRNA for ACC oxidase D67038 Solanumtuberosum 1-aminocyclopropane-1-carboxylate AF384821 oxidase (ACO2) mRNAVigna radiata ACC oxidase gene, complete cds AF315316

The genes of the 20 ACC-oxidase genes were compared and analyzed usingboth ClustalW and MAP for multiple alignment. DNAStar package (LaserGeneSoftware, Madison, Wis.) was used for editing and aligning, and DNAStarand the Blast 2 Sequences from NCBI were used to compare sequences bypairs. To compare the sequence of interest with the GenBank database theBlast program was used (Altschul et al., “Gapped BLAST and PSI-BLAST: ANew Generation of Protein Database Search Programs, Nucleic AcidsResearch 35:3389-3402 (1997), which is hereby incorporated by referencein its entirety). The similarity among the 20 sequences was determinedto be 73.1 -97.7% (range: 24.2%). The similarity with the identifiedconsensus sequence was determined to be 77.3-90.0% (range: 12.7%). UsingFormula I supra, it was estimated that approximately 31 nucleotideswould need to be modified to create a suitable synthetic gene. A single260 nt synthetic gene shown below, was designed using the region of highhomology within the identified consensus sequence for all the startingACC sequences, as seen in FIG. 17. FIG. 18 shows the alignmentdetermined for the 20 starting sequences compared to the syntheticsequence. The similarity with the synthetic gene was determined to be91.8-93.1% (range: 1.3%) for all of the starting genes. The modifiednucleotide for this trait, even though derived from a variety of plantspecies, has an improved homology over that of the consensus sequencecompared to the starting sequences, and a significantly improved rangeof similarity. In accordance with the present invention, this syntheticgene can then be cloned into an appropriate vector, using methodsdescribed above, and then used to transform a plurality of differentplants, plant cells or plant lines, chosen from among the differentplant species from which the starting genes were selected. The transgenecan be engineered by creating a sequence having inverted repeats thatlead to the production of a dsRNA upon transcription in the transgenicplant. Without being bound to a theory, a transgene engineered in thisway should lead to gene silencing of the endogenous gene, and, in thiscase, disruption of color development in any plant transformed with thesynthetic transgene.

This synthetic modified multi-species ACC-oxidase nucleic acid moleculehas a nucleotide sequence of SEQ ID NO: 68 as follows: ccagagctgatcaagggcct tcgggctcac acagatgctg 60 gtggcatcat cctgctgttc caagatgacaaggtcagtgg tctccagctt ctcaaagatg 120 gtgattggat tgatgttcct ccaatgaaccactccattgt catcaatctt ggtgaccagc 180 ttgaggtgat taccaatgga aaatacaagagtgtgatgca ccgtgtgatt gctcagacag 240 atggaaacag aatgtcaata gcatcgttctacaatccggg 260

Example 18 A Synthetic Sequence To Target For Degradation All KnownIsolates of A Single Viral Pathogen Using PRSV CP

A synthetic sequence able to target for degradation of all knownisolates of PRSV was generated. These isolates come from the Americas,Asia, and the Pacific (i.e., Australia and Hawaii). The desired trait isPRSV resistance in papaya (and probably some cucurbits). Sequences from47 American isolates, 29 Asian isolates, and 8 from Hawaii andAustralia, were used for a total of 84 sequences. These isolates andtheir accession numbers are shown in Table 18. It has been shown thattransgenic papayas are resistant, in most cases, to closely similarstrains of the virus (homologous resistance), which creates theunforeseen disadvantage of a potential low durability of transgenicresistance due to mutation or introduction of new variants of PRSV(Tennant et al., “Papaya Ringspot Virus Resistance of Transgenic Rainbowand Sunup is Affected By Gene Dosage, Plant Development, and CoatProtein Homology,” European J. of Plant Pathology 107:645-653 (2001),which is hereby incorporated by reference in its entirety). On the otherhand, due to an extreme dependence on sequence similarity, differenttransgenic lines should, theoretically, be created for everygeographical location in which a different variant of the virus isprevalent to keep the virus under control. Such a task, althoughapparently pragmatic, would be impractical in terms of cost and labor.Furthermore, many isolates of PRSV have been a source of CP genes thathave been already cloned, and most of them sequenced. TABLE 18 GenBankGeographical Origin Accession Country Isolate Source Number AustraliaBridgeman Do. GenBank U14736 Bundaberg GenBank U14737 W GenBank S89893W, Gatton GenBank U14739 W, Nort. Terr. GenBank U14744 P, no nameGenBank U14738 Wellington Po. GenBank U14740 Brazil No name GonsalvesLab Not applicable Bahia GenBank AF344641 Brasilia GenBank AF344650W-Brasilia GenBank AF344649 Ceara GenBank AF344647 W-Ceara GenBankAF344648 Espirito Santo GenBank AF344644 Itabela 1 GenBank AF344639Itabela 2 GenBank AF344640 Paraiba GenBank AF344645 Parana GenBankAF344643 Pernambuco GenBank AF344646 Sao Paulo GenBank AF344642 ChinaSevere GenBank X96538 Vb GenBank AF243496 India AP, partial GenBankAF323637 Bangalore GenBank AF120270 Chiengmai-1 GenBank AY010719Chiengmai-2 GenBank AY010720 Ratchaburi GenBank AY010721 UP, partialGenBank AF323638 W GenBank AF063221 P, no name GenBank AF063220Indonesia 1 GenBank AF374864 2 GenBank AF374865 Jamaica No nameGonsalves Not applicable Japan (Hanada's) GenBank E12704 (Maoka's)GenBank AB044339 Okinawa GenBank D50591 S, no name Malaysia (Maoka's)GenBank AB044342 Mexico Chiapas-11 GenBank AJ012650 Chiapas-30 GenBankAY017190 Chiapas-39 GenBank AF319500 Chiapas-40 GenBank AF319501 ColimaGenBank AF309968 Guerrero-9 GenBank AY017189 Jalisco-13 GenBank AF319482Jalisco-14 GenBank AF319483 Jalisco-39 GenBank AF319484 Michoacan-18GenBank AF319485 Michoacan-57 GenBank AF319486 Nayarit-22 GenBankAF319487 Oaxaca-27 GenBank AF319488 Oaxaca-66 GenBank AF319489 Oaxaca-80GenBank AF319490 Quintana Roo-1 GenBank AF319491 Quintana Roo-2 GenBankAF319492 Quintana Roo-3 GenBank AF319493 San Luis Potosi GenBankAF319502 Tabasco-42 GenBank AF319503 Tabasco-43 GenBank AF319504Tamaulipas-25 GenBank AF319494 Tamaulipas-70 GenBank AF319495 Veracruz-1GenBank AF319497 Veracruz-6 GenBank AJ012649 Veracruz-7 GenBank AF319507Veracruz-15 GenBank AF319506 Veracruz-18 GenBank AF319498 Veracruz-Al-18GenBank AF319496 Veracruz-27 GenBank AF319505 Veracruz-28 GenBankAJ012099 Yucatan GenBank AF319499 Sri Lanka W, no name GenBank U14741Taiwan (Maoka's) GenBank AB044340 W-Chiayi GenBank AY027810 W-PintungGenBank AY027811 W-Tainan GenBank AY027812 YK GenBank X78557 YK,complete GenBank X97251 Thailand No name Gonsalves Not applicableBangkok GenBank AY010712 Chonburi-1 GenBank AY010715 Chonburi-2 GenBankAY010716 Chonburi-3 GenBank AF405529 Chumporn GenBank AY010713 Khon KaenGenBank AY010714 Nan GenBank AF405530 Nakhon Phat. GenBank AF405532Nakhon Ratc. GenBank AF405531 Lab. Mild GenBank AY010717 Lab. SevereGenBank AY010718 (Maoka's) GenBank AB044340 W, complete GenBankNC_002814 AY010722 W, partial GenBank U14743 United States FloridaGenBank AF196839 Hawaii, HA GenBank X67673 Idem, complete GenBank S46722Hawaii, revised GenBank NC_001785 Hawaii, Kapoho Gonsalves Notapplicable Hawaii, Keaau Gonsalves Not applicable Hawaii, Oahu GonsalvesNot applicable Puerto Rico GenBank AF196838 Venezuela El Vigia GonsalvesNot applicable Lagunillas Gonsalves Not applicable Vietnam W, no nameGenBank U14742

Eighty-four sequences of geographical isolates of PRSV worldwide wereanalyzed and compared by multiple alignments for the CP gene alone.Given that CP gene sequences have been obtained from differentlaboratories using different cloning strategies, it was necessary totrim the sequences in order to compare only the region that wasavailable for (and shared by) all of them. After this step, thesequences were once again aligned. DNAStar package was used for editingand aligning, and DNAStar and the Blast 2 Sequences from NCBI were usedto compare sequences by pairs. The similarity among the startingsequences was determined to be 79.2-100% (range: 20.8%). The similaritywith the consensus was found to be 84.3-99.5% (range: 15.2%).

After the sequences were aligned, a potential location of high homologyamenable to manipulation was chosen. Using Formula I, supra, it wasdetermined that approximately 20 nucleotides would need to bemanipulated to create a suitable synthetic gene. A reference sequencewas chosen and nucleotides modified until a 216 nt nucleic acid moleculewas determined to be suitable for gene silencing of all 84 PRSVisolates. FIG. 19 shows the CLUSTALW alignment of the resultingsynthetic “Universal” PRSV isolate sequence. Sequence similarityanalysis with the synthetic gene was 89.8-93.5% (range: 3.7%) comparedto all starting sequences. The homology of the synthetic sequencerelative to the starting sequences, was lower than the maximum homologyof the starting sequences, but was above the lower limits of similarityfound for the consensus sequence relative to the starting sequences. Thesynthetic nucleic acid also had a narrow range of variation insimilarity compared to all starting sequences (i.e., 0.8%) compared to arange of 10.5% variation in similarity among the native startingsequences. This combination of homology greater than 80% and a narrowrange of variation make the Petunia synthetic nucleic acid molecule agood candidate for degrading RNA in a target host because thetranscribed dsRNA is less likely to be perceived as foreign by its host,and will therefore be able to initiate RNA degradation of the targethost RNA and impart its intended trait. This universal PRSV syntheticmolecule has a nucleotide sequence of SEQ ID NO: 156 as follows:gccagatacg ctttcgattt ctatgaggtg aattcaaaaa 60 cacctgatag agctcgtgaagctcacacgc agatgaaagc tgcagcactg cgtaacacta 120 atcgcagaat gtttggaatggacggcagtg tcagtaacaa agaagaaaac acagaaagac 180 acacagtgga agatgtaaacagagacatgc actctctcct gggtatgcgc aactga 216

Additionally, the CP genes were analyzed in smaller geographicgroupings. All isolates from North and South American countries(excluding those from Hawaii) were grouped as “the Americas.” Thestarting sequences within this group were aligned using CLUSTALW, andDNAStar and the Blast 2 Sequences from NCBI were used to comparesequences by pairs. This analysis showed a similarity among startingsequences of 91.7-100% (range: 8.3%), with a similarity of 94-100%(range: 6.0%) for the starting sequences compared to the consensussequence. A reference sequence was chosen from a region of high homologyand select nucleotides were manipulated. Using Formula I, supra, it wasdetermined that approximately 20 nucleotides would need to bemanipulated to create a suitable synthetic gene. The modified sequencewas aligned with the starting sequences numerous times until a modified216 nt synthetic “universal” nucleic acid molecule for the combined“Americas” isolates was determined to be suitable. The final CLUSTALWalignment is shown in FIG. 20. The final homology analysis resulted in asimilarity of 93.1-94.9% (range: 1.8%) with the synthetic gene comparedto all starting sequences. The modified sequence had a degree ofhomology over 90%, with a much improved range of variation in similarityvalues over those of the starting sequences or even the consensussequence. The “universal Americas” PRSV synthetic molecule has anucleotide sequence of SEQ ID NO: 153 as follows: gccagatacg cttttgatttctatgaggtg aattcaaaaa 60 cacctgatag agctcgtgaa gctcacatgc agatgaaggctgcagcgctg cgaaacacta 120 atcgtagaat gtttggtatg gacggcagtg ttagcaacaatgaagaaaac acggagagac 180 acacagtgga agatgtcaat agagacatgc actctctcctgggtatgcgc aactaa 216

All isolates from Asia were grouped as “Asia.” The starting sequenceswithin this group were aligned using CLUSTALW, and DNAStar and the Blast2 Sequences from NCBI were used to compare sequences by pairs. Thisanalysis showed a similarity of 80.6-99.5% (range: 18.9%) among startingsequences and a similarity of 84.3-99.5% (range: 15.2%) for the startingsequences compared to the consensus sequence. A reference sequence waschosen from a region of high homology and select nucleotides weremanipulated as described above. Using Formula I, supra, it wasdetermined that approximately 20 nucleotides would need to bemanipulated to create a suitable synthetic gene. The modified sequenceswere aligned numerous times until a final 216 nt synthetic “universal”nucleic acid molecule for the combined “Asia” isolates was determined tobe suitable. The final CLUSTALW alignment is shown in FIG. 21. The finalhomology analysis showed a similarity of 89.9-93.1% (range: 3.2%) withthe synthetic gene compared to all starting sequences. The modified Asiasequence has a fairly high degree of homology, well over the 80%minimum. Striking here is the variation in the range of similarity,which was improved significantly from that among the starting sequencesthemselves (18.9%). While the modifications in this trait group werehalted when the values shown here were reached, it is highly probablethat the 3.2% variance in homology can be further reduced withadditional manipulations. This “universal Asia” PRSV synthetic moleculehas a nucleotide sequence of SEQ ID NO: 154 as follows: gccagatatgctttcgattt ctatgaagtg aattcaaaaa 60 cacctgatag agctcgtgaa gctcacatgcagatgaaagc tgcagcactg cgtaacgcta 120 atcgcagaat gtttggaatg gacggcactgtcagtaacaa agaagaaaac acagaaagac 180 acacagtgga agatgtaaac agagacatgcaatctctcct gggtatgcgc aactga 216

All isolates from Australia and Hawaii were grouped as the “Pacific”isolates. The sequences within each of these groups were aligned usingthe CLUSTALW, and DNAStar and the Blast 2 Sequences from NCBI were usedto compare sequences by pairs. This analysis showed a similarity amongstarting sequences of 96.8-99.5% (range: 2.7%), with the similarity of96.8-100% (range: 2.7%) for the starting sequences compared to theconsensus sequence. Following alignment, a potential location amenableto manipulation was chosen. In this case, due to the high similarityvalue among the starting sequences (96.8-99.5%), only about 4 nts arerequired for manipulation in order to create a suitable modifiedsynthetic nucleic acid molecule. A reference sequence was selected,manipulated, and aligned, and a modified 216 nt synthetic “universal”nucleic acid molecule was determined to be suitable for gene silencingof PRSV isolates within the Pacific geographic group. The final CLUSTALWalignment is shown in FIG. 22. The similarity with the synthetic genewas determined to be 97.7-98.1% (range: 0.4%) compared to all startingsequences. The modified sequence of this trait group has a high homologyvalue that is very similar to each of the starting sequences (0.4%variation), which is an improvement over the native starting sequencesthemselves, and in comparison to the consensus sequence. This traitgroup is an example that the present invention can create a moleculewith improved range of variation in similarity values, even from a traitgroup which exhibits high native homology. This “universal Pacific” PRSVsynthetic molecule has a nucleotide sequence of SEQ ID NO: 151 asfollows: gctagatatg ctttcgattt ttatgaggtg aattcgaaaa 60 cacctgatagggctcgcgaa gctcacatgc agatgaaagc tgcagcgctg cgaaacacta 120 gtcgcagaatgtttggtatg gacggcagtg ttagtaacaa ggaagaaaac acggagagac 180 acacagtggaagatgtcaat agagacatgc actctctcct gggtatgcgc aactaa 216

All of the “universal” synthetic sequences of the present invention canbe used to prepare DNA constructs of the present invention, as describedabove, that are capable of producing transcribed dsRNA of the nucleicacid sequence in the plant, plant cell or plant line transformed withthe construct, thereby effecting gene silencing and imparting the traitof resistance to multiple strains of PRSV, a devastating pathogen ofCarica papaya worldwide.

Fusing multiple synthetic modified genes in a single transgene mayresult in a single construct able to trigger gene silencing andresistance against all isolates of PRSV. For example, the Universalsynthetic nucleic acid molecule could be combined in a single constructwith the synthetic molecule for the Americas; or all three universalgeographic synthetic sequences could be combined in a single transgene.With the PCR assembly strategy (Stemmer et al., “Single-Step Assembly ofa Gene and Entire Plasmid From Large Numbers ofOligodeoxyribonucleotides,” Gene 164:49-53 (1985), which is herebyincorporated by reference in its entirety) that was already successfullyused with the N gene of different tospoviruses as explained above, ithighly likely that such a transgene would be effective.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A DNA construct comprising: a modified nucleic acid molecule having anucleotide sequence which is at least 80%, but less than 100%,homologous to two or more desired trait DNA molecules from a viral plantpathogen and which achieves post-transcriptional gene silencing of thehomologous desired trait DNA molecules, thereby imparting the desiredtrait to plants transformed with said DNA construct, wherein each of thedesired trait DNA molecules relative to the modified nucleic acidmolecule have a nucleotide sequence similarity value and each of thesesimilarity values differs by no more than 3 percentage points, andwherein the desired trait is viral resistance in plants, wherein themodified nucleic acid has a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 30,SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 154, and SEQ ID NO:
 156. 2.The DNA construct according to claim 1 further comprising: a promotersequence and a termination sequence, wherein the promoter sequence andthe termination sequence are operatively coupled to said modifiednucleic acid molecule.
 3. The DNA construct according to claim 1,wherein said modified nucleic acid molecule encodes an RNA moleculewhich is translatable.
 4. The DNA construct according to claim 1,wherein said modified nucleic acid molecule encodes an RNA moleculewhich is non-translatable.
 5. The DNA construct according to claim 1,wherein each of the desired trait DNA molecules relative to the modifiednucleic acid molecule have a nucleotide sequence similarity value andeach of these similarity values differs by no more than 2 percentagepoints.
 6. The DNA construct according to claim 1, wherein each of thedesired trait DNA molecules relative to the modified nucleic acidmolecule have a nucleotide sequence similarity value and each of thesesimilarity values differs by 1 percentage point or less.
 7. A DNAexpression vector comprising the DNA construct according to claim
 1. 8.A host cell transformed with the DNA construct of claim 1, wherein thecell is a bacterial cell or a plant cell.
 9. The host cell according toclaim 8, wherein the cell is a plant cell.
 10. A transgenic planttransformed with the DNA construct according to claim
 1. 11. Thetransgenic plant according to claim 10, wherein the modified nucleicacid molecule has a nucleotide sequence which is at least 80%, but lessthan 100%, homologous to a plurality of desired trait DNA molecules,wherein each of the desired trait DNA molecules relative to the modifiednucleic acid molecule have a nucleotide sequence similarity value andeach of these similarity values differs by no more than 2 percentagepoints.
 12. The transgenic plant according to claim 10 furthercomprising: a promoter sequence and a termination sequence, wherein thepromoter sequence and the termination sequence are operatively coupledto said modified nucleic acid.
 13. The transgenic plant according toclaim 10, wherein the plant is papaya.
 14. A transgenic plant seedtransformed with the DNA construct according to claim
 1. 15. Thetransgenic plant seed according to claim 14, wherein said modifiednucleic acid molecule has a nucleotide sequence which is at least 80%,but less than 100%, homologous to a plurality of desired trait DNAmolecules, wherein each of the desired trait DNA molecules relative tothe modified nucleic acid molecule have a nucleotide sequence similarityvalue and each of these similarity values differs by 2 percentage pointsor less.
 16. The transgenic plant seed according to claim 15, whereinthe plant is papaya.
 17. A DNA construct comprising: a plurality ofmodified nucleic acid molecules, wherein each modified nucleic acidmolecule has a nucleotide sequence which is at least 80%, but less than100%, homologous to one or more desired trait DNA molecules from a viralplant pathogen, wherein said plurality of modified nucleic acidmolecules achieves post-transcriptional gene silencing of the homologousdesired trait DNA molecules, thereby collectively imparting a pluralityof traits to plants transformed with said DNA construct, and wherein thedesired trait DNA molecules relative to their respective modifiednucleic acid molecule have a nucleotide sequence similarity value andeach of these similarity values differs by no more than 3 percentagepoints, and wherein the desired traits are viral resistance in plants,wherein the modified first nucleic acid has a nucleotide sequence isselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 9, SEQ IDNO: 23, SEQ ID NO: 30,SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 154,and SEQ ID NO:
 156. 18. The DNA construct according to claim 17, whereinthe desired trait DNA molecules relative to their respective modifiednucleic acid molecule have a nucleotide sequence similarity value andeach of these similarity values differs by no more than 2 percentagepoints.
 19. The DNA construct according to claim 17, wherein the desiredtrait DNA molecules relative to their respective modified nucleic acidmolecule have a nucleotide sequence similarity value and each of thesesimilarity values differs by no more than 1 percentage point.
 20. TheDNA construct according to claim 17 further comprising: a promotersequence and a termination sequence, wherein the promoter sequence andthe termination sequence are operatively coupled to said plurality ofmodified nucleic acid molecules.
 21. The DNA construct according toclaim 17, wherein at least some of said modified nucleic acid moleculesencode RNA molecules which are translatable.
 22. The DNA constructaccording to claim 17, wherein at least some of said modified nucleicacid molecules encode RNA molecules which are nontranslatable.
 23. TheDNA construct according to claim 17, wherein said DNA construct effectspost-transcriptional gene silencing within plants.
 24. An expressionvector comprising the DNA construct according to claim
 17. 25. A hostcell transformed with the DNA construct according to claim 17, whereinthe cell is a bacterial cell or a plant cell.
 26. The host cellaccording to claim 25, wherein the cell is a plant cell.
 27. Atransgenic plant transformed with the DNA construct of claim
 17. 28. Thetransgenic plant according to claim 27, wherein the modified nucleicacid molecules have a nucleotide sequence which is at least 80%, butless than 100%, homologous to one or more desired trait DNA moleculesand wherein each of the desired trait DNA molecules relative to theirrespective modified nucleic acid molecule have a nucleotide sequencesimilarity value and each of these similarity values differs by no morethan 2 percentage points.
 29. The transgenic plant according to claim 27further comprising: a promoter sequence and a termination sequence,wherein the promoter sequence and the termination sequence areoperatively coupled to the plurality of modified nucleic acid molecules.30. The transgenic plant according to claim 27, wherein at least some ofsaid modified nucleic acid molecules encode RNA molecules which aretranslatable.
 31. The transgenic plant according to claim 27, wherein atleast some of said modified nucleic acid molecules encode RNA moleculeswhich are non-translatable.
 32. The transgenic plant according to claim27, wherein the plant is papaya.
 33. A transgenic plant seed transformedwith the DNA construct according to claim
 17. 34. The transgenic plantseed according to claim 33, wherein each of the modified nucleic acidmolecules have a nucleotide sequence which is at least 80%, but lessthan 100%, homologous to one or more of desired trait DNA molecules andwherein each of the desired trait DNA molecules relative to theirrespective modified nucleic acid molecule have a nucleotide sequencesimilarity value and each of these similarity values differs by no morethan 2 percentage points.
 35. The transgenic plant seed according toclaim 33, wherein the plant is papaya.
 36. A method of imparting a traitto plants comprising: transforming a plant with the DNA constructaccording to claim
 1. 37. The method according to claim 36, wherein eachof the desired trait DNA molecules relative to the modified nucleic acidmolecule have a nucleotide sequence similarity value and each of thesesimilarity values differs by no more than 2 percentage points.
 38. Themethod according to claim 36, wherein each of the desired trait DNAmolecules relative to the modified nucleic acid molecule has anucleotide sequence similarity value which differs by no more than 1percentage point.
 39. The method according to claim 36 furthercomprising: propagating progeny of the transgenic plant.
 40. A method ofimparting a trait to plants comprising: planting the transgenic plantseed according to claim 14 and propagating a plant from the plantedtransgenic plant seed.
 41. A method of imparting one or more traits toplants comprising: transforming a plant with the DNA construct accordingto claim
 17. 42. The method according to claim 41, wherein said modifiednucleic acid molecules have a nucleotide sequence which is at least 80%,but less than 100%, homologous to one or more desired trait DNAmolecules, and wherein each of the desired trait DNA molecules relativeto the modified nucleic acid molecule have a nucleotide sequencesimilarity value and each of these similarity values differs by no morethan 2 percentage points.
 43. The method according to claim 41 furthercomprising: propagating progeny of the transgenic plants.
 44. A methodof imparting a trait to plants comprising: planting the transgenic plantseed according to claim 33 and propagating a plant from the plantedtransgenic plant seed.
 45. A DNA construct comprising a first nucleicacid molecule having a nucleotide sequence which is at least 80%, butless than 100%, homologous to two or more desired trait DNA moleculesfrom a viral plant pathogen, and which has a length that is insufficientto independently impart a desired trait to plants transformed with saidDNA construct, wherein each of the desired trait DNA molecules relativeto the first nucleic acid molecule have a nucleotide sequence similarityvalue and each of these similarity values differs by no more than 3percentage points, and wherein the desired trait is viral resistance inplants; and a second DNA molecule, operatively coupled to the firstnucleic acid molecule, wherein the second DNA molecule has a length thatis sufficient to achieve post-transcriptional silencing of thehomologous desired trait DNA molecules, wherein said first nucleic acidmolecule and said second DNA molecule collectively achievepost-transcriptional silencing, thereby imparting the desired trait toplants transformed with said DNA construct, wherein the first nucleicacid has a nucleotide sequence selected from the group consisting of SEQID NO: 5, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 151,SEQ ID NO: 153, SEQ ID NO: 154, and SEQ ID NO:
 156. 46. The DNAconstruct according to claim 45 further comprising: a promoter sequenceand a termination sequence, wherein the promoter sequence and thetermination sequence are operatively coupled to the first and the secondDNA molecules.
 47. The DNA construct according to claim 45, wherein eachof the desired trait DNA molecules relative to the modified nucleic acidmolecule has a nucleotide sequence similarity value and each of thesesimilarity values differs by 1 percentage point or less.
 48. The DNAconstruct according to claim 45, further comprising: a plurality ofmodified nucleic acid molecules, each modified nucleic acid moleculebeing directed to a different trait than the other modified nucleic acidmolecules in the DNA construct.
 49. The DNA construct according to claim48, wherein the modified nucleic acid molecules have a nucleotidesequence which is at least 80%, but less than 100%, homologous to aplurality of desired trait DNA molecules, and wherein the desired traitDNA molecules relative to the respective modified nucleic acid moleculehave a nucleotide sequence similarity value and each of these similarityvalues differs by no more than 3 percentage points.
 50. A DNA expressionvector comprising the DNA construct according to claim
 45. 51. A hostcell transformed with the DNA construct according to claim 45, whereinthe cell is a bacterial cell or a plant cell.
 52. The host cellaccording to claim 51, wherein the cell is a plant cell.
 53. Atransgenic plant transformed with the DNA construct according to claim45.
 54. The transgenic plant according to claim 53, wherein the plant ispapaya.
 55. A transgenic plant seed transformed with the DNA constructaccording to claim
 45. 56. The transgenic plant seed according to claim55, wherein the plant is papaya.
 57. A method of imparting a trait toplants comprising: transforming a plant with the DNA construct accordingto claim
 45. 58. The method according to claim 57, wherein each of thedesired trait DNA molecules relative to the modified nucleic acidmolecule have a nucleotide sequence similarity value and each of thesesimilarity values differs by no more than 2 percentage points.
 59. Themethod according to claim 57, wherein each of the desired trait DNAmolecules relative to the modified nucleic acid molecule have anucleotide sequence similarity value and each of these similarity valuesdiffers by no more than 1 percentage point.
 60. The DNA constructaccording to claim 1, further comprising a second DNA molecule coupledto the first DNA molecule, wherein the first DNA molecule and the secondDNA molecule achieve post-transcriptional silencing of the homologousdesired trait DNA molecules, and impart the desired trait to plantstransformed with said DNA construct.
 61. The DNA construct according toclaim 60 further comprising: a promoter sequence and a terminationsequence, wherein the promoter sequence and the termination sequence areoperatively coupled to the first and the second DNA molecules.